Abstract
Elevated expression of N-acetyltransferase 1 (NAT1) is associated with invasive and lobular breast carcinomas as well as with bone metastasis following an epithelial-to-mesenchymal transition. We investigated the effect of NAT1 gene deletion in three different human breast cancer cell lines, MDA-MB-231, MCF-7, and ZR-75-1. Human NAT1 was knocked out using CRISPR/Cas9 technology and two different guide RNAs. None of the NAT1 knockout (KO) cell lines exhibited detectable NAT1 activity when measured using its selective substrate p-aminobenzoic acid (PABA). Endogenous acetyl coenzyme A levels (cofactor for acetylation pathways) in NAT1 KO cell lines were significantly elevated in the MDA-MB-231 (p < 0.001) and MCF-7 (p=0.0127) but not the ZR-75-1 (p > 0.05). Although the effects of NAT1 KO on cell-doubling time were inconsistent across the three breast cancer cell lines, the ability of the NAT1 KO cell lines to form anchorage-independent colonies in soft agar was dramatically and consistently reduced in each of the breast cancer cell lines. The NAT1 KO clones for MDA-MB-231, MCF-7, and ZR-75-1 had a reduction greater than 20-, 6-, and 7- folds in anchorage-independent cell growth, respectively, compared to their parental cell lines (p < 0.0001, p < 0.0001, and p < 0.05, respectively). The results indicate that NAT1 may be an important regulator of cellular acetyl coenzyme A levels and strongly suggest that elevated NAT1 expression in breast cancers contribute to their anchorage-independent growth properties and ultimately metastatic potential.
Highlights
Human arylamine N-acetyltransferase 1 (NAT1) catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to arylamine and hydrazine substrates [1, 2]
A recent report demonstrated that congenic rats expressing high levels of rat N-acetyltransferase 2 (NAT2; ortholog to human NAT1) activity exhibited more mammary tumors, and this finding was independent of carcinogen metabolism [12]. e effects of inhibition or overexpression of human NAT1 has been the focus of previous studies [13,14,15,16,17]
MDA-MB-231 cells with increased NAT1 activity showed lower endogenous AcCoA levels, compared to the parental cell line [17]. ese observations, together with the wide-spread tissue distribution of NAT1 and its presence in almost all species [18] and its ability to catalyze the hydrolysis of AcCoA, suggest the role of NAT1 in carcinogenesis might be related to the regulation of AcCoA
Summary
Human arylamine N-acetyltransferase 1 (NAT1) catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to arylamine and hydrazine substrates [1, 2]. Human NAT1 catalyzes hydrolysis of AcCoA in the presence of folate [3, 4]. Additional studies have reported elevated NAT1 expression in estrogen receptor-positive tumors [7,8,9] as well as with bone metastasis following an epithelial-tomesenchymal transition (EMT) [10, 11]. A recent report demonstrated that congenic rats expressing high levels of rat N-acetyltransferase 2 (NAT2; ortholog to human NAT1) activity exhibited more mammary tumors, and this finding was independent of carcinogen metabolism [12]. MDA-MB-231 cells with increased NAT1 activity showed lower endogenous AcCoA levels, compared to the parental cell line [17]. ese observations, together with the wide-spread tissue distribution of NAT1 and its presence in almost all species [18] and its ability to catalyze the hydrolysis of AcCoA, suggest the role of NAT1 in carcinogenesis might be related to the regulation of AcCoA
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