Deletion of arylamine N-acetyltransferase 1 in MDA-MB-231 human breast cancer cells reduces primary and secondary tumor growth in vivo with no significant effects on metastasis.

Arylamine N-acetyltransferase 1 (NAT1) is frequently upregulated in breast cancer. Previous studies showed that inhibition or depletion of NAT1 in breast cancer cells diminishes anchorage-independent growth in culture, suggesting that NAT1 contributes to breast cancer growth and metastasis. To further investigate the contribution of NAT1 to growth and cell invasive/migratory behavior, we subjected parental and NAT1 knockout (KO) breast cancer cell lines (MDA-MB-231, MCF-7, and ZR-75-1) to multiple assays. The rate of cell growth in suspension was not consistently decreased in NAT1 KO cells across the cell lines tested. Similarly, cell migration and invasion assays failed to produce reproducible differences between the parental and NAT1 KO cells. To overcome the limitations of in vitro assays, we tested parental and NAT1 KO cells in vivo in a xenograft model by injecting cells into the flank of immunocompromised mice. NAT1 KO MDA-MB-231 cells produced primary tumors smaller than those formed by parental cells, which was contributed by an increased rate of apoptosis in KO cells. The frequency of lung metastasis, however, was not altered in NAT1 KO cells. When the primary tumors of the parental and NAT1 KO cells were allowed to grow to a pre-determined size or delivered directly via tail vein, the number and size of metastatic foci in the lung did not differ between the parental and NAT1 KO cells. In conclusion, NAT1 contributes to primary and secondary tumor growth in vivo in MDA-MB-231 breast cancer cells but does not appear to affect its metastatic potential.

Identification and characterization of potent, selective, and efficacious inhibitors of human arylamine N-acetyltransferase 1.

Arylamine N-acetyltransferase 1 (NAT1) plays a pivotal role in the metabolism of carcinogens and is a drug target for cancer prevention and/or treatment. A protein-ligand virtual screening of 2 million chemicals was ranked for predicted binding affinity towards the inhibition of human NAT1. Sixty of the five hundred top-ranked compounds were tested experimentally for inhibition of recombinant human NAT1 and N-acetyltransferase 2 (NAT2). The most promising compound 9,10-dihydro-9,10-dioxo-1,2-anthracenediyl diethyl ester (compound 10) was found to be a potent and selective NAT1 inhibitor with an in vitro IC50 of 0.75 µM. Two structural analogs of this compound were selective but less potent for inhibition of NAT1 whereas a third structural analog 1,2-dihydroxyanthraquinone (a compound 10 hydrolysis product also known as Alizarin) showed comparable potency and efficacy for human NAT1 inhibition. Compound 10 inhibited N-acetylation of the arylamine carcinogen 4-aminobiphenyl (ABP) both in vitro and in DNA repair-deficient Chinese hamster ovary (CHO) cells in situ stably expressing human NAT1 and CYP1A1. Compound 10 and Alizarin effectively inhibited NAT1 in cryopreserved human hepatocytes whereas inhibition of NAT2 was not observed. Compound 10 caused concentration-dependent reductions in DNA adduct formation and DNA double-strand breaks following metabolism of aromatic amine carcinogens beta-naphthylamine and/or ABP in CHO cells. Compound 10 inhibited proliferation and invasion in human breast cancer cells and showed selectivity towards tumorigenic versus non-tumorigenic cells. In conclusion, our study identifies potent, selective, and efficacious inhibitors of human NAT1. Alizarin's ability to inhibit NAT1 could reduce breast cancer metastasis particularly to bone.

Knockout of human arylamine N-acetyltransferase 1 (NAT1) in MDA-MB-231 breast cancer cells leads to increased reserve capacity, maximum mitochondrial capacity, and glycolytic reserve capacity.

Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. NAT1 can also hydrolyze acetyl-coenzyme A (acetyl-CoA) in the absence of an arylamine substrate. Expression of NAT1 varies between individuals and is elevated in several cancers including estrogen receptor positive (ER+) breast cancers. To date, however, the exact mechanism by which NAT1 expression affects mitochondrial bioenergetics in breast cancer cells has not been described. To further evaluate the role of NAT1 in energy metabolism MDA-MB-231 breast cancer cells with parental, increased, and knockout levels of NAT1 activity were compared for bioenergetics profile. Basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured followed by programmed sequential injection of Oligomycin (ATP synthase inhibitor), FCCP (ETC uncoupler), Antimycin A (Complex III inhibitor), and Rotenone (Complex I inhibitor) to evaluate mitochondrial bioenergetics. Compared to the cell lines with parental NAT1 activity, NAT1 knockout MDA-MB-231 cell lines exhibited significant differences in bioenergetics profile, while those with increased NAT1 did not. Significant increases in reserve capacity, maximum mitochondrial capacity, and glycolytic reserve capacity were observed in NAT1 knockout MDA-MB-231 cell lines compared to those with parental and increased NAT1 activity. These data indicate that NAT1 knockout in MDA-MB-231 breast cancer cells may enhance adaptation to stress by increasing plasticity in response to energy demand.

Genetic and small molecule inhibition of arylamine N-acetyltransferase 1 reduces anchorage-independent growth in human breast cancer cell line MDA-MB-231.

Arylamine N-acetyltransferase 1 (NAT1) expression is reported to affect proliferation, invasiveness, and growth of cancer cells. MDA-MB-231 breast cancer cells were engineered such that NAT1 expression was elevated or suppressed, or treated with a small molecule inhibitor of NAT1. The MDA-MB-231 human breast cancer cell lines were engineered with a scrambled shRNA, a NAT1 specific shRNA or a NAT1 overexpression cassette stably integrated into a single flippase recognition target (FRT) site facilitating incorporation of these different genetic elements into the same genomic location. NAT1-specific shRNA reduced NAT1 activity in vitro by 39%, increased endogenous acetyl coenzyme A levels by 35%, and reduced anchorage-independent growth (sevenfold) without significant effects on cell morphology, growth rates, anchorage-dependent colony formation, or invasiveness compared to the scrambled shRNA cell line. Despite 12-fold overexpression of NAT1 activity in the NAT1 overexpression cassette transfected MDA-MB-231 cell line, doubling time, anchorage-dependent cell growth, anchorage-independent cell growth, and relative invasiveness were not changed significantly when compared to the scrambled shRNA cell line. A small molecule (5E)-[5-(4-hydroxy-3,5-diiodobenzylidene)-2-thioxo-1,3-thiazolidin-4-one (5-HDST) was 25-fold more selective towards the inhibition of recombinant human NAT1 than N-acetyltransferase 2. Incubation of MDA-MB-231 cell line with 5-HDST resulted in 60% reduction in NAT1 activity and significant decreases in cell growth, anchorage-dependent growth, and anchorage-independent growth. In summary, inhibition of NAT1 activity by either shRNA or 5-HDST reduced anchorage-independent growth in the MDA-MB-231 human breast cancer cell line. These findings suggest that human NAT1 could serve as a target for the prevention and/or treatment of breast cancer.

Arylamine N-acetyltransferase 1 in situ N-acetylation on CD3+ peripheral blood mononuclear cells correlate with NATb mRNA and NAT1 haplotype.

Human arylamine N-acetyltransferase 1 (NAT1) is responsible for the activation and elimination of xenobiotic compounds and carcinogens. Genetic polymorphisms in NAT1 modify both drug efficacy and toxicity. Previous studies have suggested a role for NAT1 in the development of several diseases. The aim of the present study was to evaluate NAT1 protein expression and in situ N-acetylation capacity in peripheral blood mononuclear cells (PBMC), as well as their possible associations with the expression of NAT1 transcript and NAT1 genotype. We report NAT1 protein, mRNA levels, and N-acetylation in situ activity for PBMC obtained from healthy donors. NAT1-specific protein expression was higher in CD3+ cells than other major immune cell subtypes (CD19 or CD56 cells). N-acetylation of pABA varied markedly among the PBMC of participants, but correlated very significantly with levels of NAT1 transcripts. NAT1*4 subjects showed significantly (p = 0.017) higher apparent pABA V max of 71.3 ± 3.7 versus the NAT1*14B subjects apparent V max of 58.5 ± 2.5 nmoles Ac-pABA/24 h/million cells. Levels of pABA N-acetylation activity at each concentration of substrate evaluated also significantly correlated with NAT1 mRNA levels for all samples (p < 0.0001). This highly significant correlation was maintained for samples with the NAT1*4 (p = 0.002) and NAT1*14B haplotypes (p = 0.0106). These results provide the first documentation that NAT1-catalyzed N-acetylation in PBMC is higher in T cell than in other immune cell subtypes and that individual variation in N-acetylation capacity is dependent upon NAT1 mRNA and NAT1 haplotype.

Congenic rats with higher arylamine N-acetyltransferase 2 activity exhibit greater carcinogen-induced mammary tumor susceptibility independent of carcinogen metabolism.

BACKGROUND: Recent investigations suggest role(s) of human arylamine N-acetyltransferase 1 (NAT1) in breast cancer. Rat NAT2 is orthologous to human NAT1 and the gene products are functional homologs. We conducted in vivo studies using F344.WKY-Nat2 rapid/slow rats, congenic at rat Nat2 for high (rapid) and low (slow) arylamine N-acetyltransferase activity, to assess a possible role for rat NAT2 in mammary tumor susceptibility. METHODS: Mammary carcinogens, methylnitrosourea (MNU) and 7,12-dimethylbenzanthracene (DMBA) neither of which is metabolized by N-acetyltransferase, were administered to assess mammary tumors. MNU was administered at 3 or 8 weeks of age. DMBA was administered at 8 weeks of age. NAT2 enzymatic activity and endogenous acetyl-coenzyme A (AcCoA) levels were measured in tissue samples and embryonic fibroblasts isolated from the congenic rats. RESULTS: Tumor latency was shorter in rapid NAT2 rats compared to slow NAT2 rats, with statistical significance for MNU administered at 3 and 8 weeks of age (p = 0.009 and 0.050, respectively). Tumor multiplicity and incidence were higher in rapid NAT2 rats compared to slow NAT2 rats administered MNU or DMBA at 8 weeks of age (MNU, p = 0.050 and 0.035; DMBA, p = 0.004 and 0.027, respectively). Recombinant rat rapid-NAT2, as well as tissue samples and embryonic fibroblasts derived from rapid NAT2 rats, catalyzed p-aminobenzoic acid N-acetyl transfer and folate-dependent acetyl-coenzyme A (AcCoA) hydrolysis at higher rates than those derived from rat slow-NAT2. Embryonic fibroblasts isolated from rapid NAT2 rats displayed lower levels of cellular AcCoA than slow NAT2 rats (p < 0.01). CONCLUSIONS: A novel role for rat NAT2 in mammary cancer was discovered unrelated to carcinogen metabolism, suggesting a role for human NAT1 in breast cancer.

Human N-Acetyltransferase 1 and 2 Differ in Affinity Towards Acetyl-Coenzyme A Cofactor and N-Hydroxy-Arylamine Carcinogens.

Arylamine N-acetyltransferases catalyze the transfer of acetyl groups from the endogenous cofactor acetyl coenzyme A (AcCoA) to arylamine (N-acetylation) and N-hydroxy-arylamine (O-acetylation) acceptors. Humans express two arylamine N-acetyltransferase isozymes (NAT1 and NAT2) which catalyze both N- and O-acetylation but differ in genetic regulation, substrate selectivity, and expression in human tissues. We investigated recombinant human NAT1 and NAT2 expressed in an Escherichia coli JM105 and Schizosaccharomyces pombe expression systems as well as in Chinese hamster ovary (CHO) cells to assess the relative affinity of AcCoA for human NAT1 and NAT2. NAT1 and NAT2 affinity for AcCoA was higher for recombinant human NAT1 than NAT2 when catalyzing N-acetylation of aromatic amine carcinogens 2-aminofluroene (AF), 4-aminobiphenyl (ABP), and ß-naphthylamine (BNA) and the metabolic activation of N-hydroxy-2-aminofluorene (N-OH-AF) and N-hydroxy-4-aminobiphenyl (N-OH-ABP) via O-acetylation. These results suggest that AcCoA level may influence differential rates of arylamine carcinogen metabolism catalyzed by NAT1 and NAT2 in human tissues. Affinity was higher for NAT2 than for NAT1 using N-OH-AF and N-OH-ABP as substrate consistent with a larger active site for NAT2. In conclusion, following recombinant expression in bacteria, yeast, and CHO cells, we report significant differences in affinity between human NAT1 and NAT2 for its required co-factor AcCoA, as well as for N-hydroxy-arylamines activated via O-acetylation. The findings provide important information to understand the relative contribution of human NAT1 vs NAT2 towards N-acetylation and O-acetylation reactions in human hepatic and extrahepatic tissues.

Arylamine N-Acetyltransferase 1 Activity is Regulated by the Protein Acetylation Status.

Arylamine N-acetyltransferase 1 (NAT1) is a drug metabolizing enzyme that influences cancer cell proliferation and survival, especially in breast cancer. Lysine-acetylation is an important Post-Translational Modification (PTM) in the regulation of diverse cellular processes. Histone deacetylases (HDACs) and Sirtuins (SIRT) may have an important role on the NAT1 acetylation status, affecting its catalytic capacity and having an impact on the downstream functions of this protein. The aim of the present work is to investigate the acetylation status of NAT1 in human breast cancer. Breast cancer cell lines MDA-MB-231 (ER-, PR-, HER2-) and ZR-75-1 (estrogen receptor+, PR+, HER2+) were cultured in the presence of HDAC inhibitors (SAHA, TSA) or Sirtuin inhibitors (AGK2, EX527, Sirtinol). Under these conditions, NAT1 protein and gene expression as well as enzymatic activity were quantified. Acetylation of NAT1 protein was evaluated following an immunoprecipitation protocol and acetyl-Lysine quantification. Sirt1 and Sirt2 knockdown were performed and NAT1 protein and NAT1 mRNA expression and catalytic activity were quantified. The treatment of MDA-MB-231 or ZR-75-1 cells with increasing HDAC inhibitors resulted in 2 to 15-fold upregulation in NAT1 message expression. Finally, the catalytic activity of NAT1 in the presence of HDAC inhibition increased 2-fold. Conversely, the inhibition of Sirtuin activity did not cause significant changes in NAT1 message but produced a significant decrease in NAT1 catalytic activity. NAT1 acetylation was higher in the cells treated with HDAC inhibitors, as well as Sirtuin inhibitors. Finally, silencing of Sirt1 and Sirt2 genes by siRNA transient knockdown of each or both genes resulted in reduction of NAT1 protein expression and catalytic activity. The use of HDAC and Sirtuin inhibitors has been demonstrated as a promising powerful therapeutic alternative in various cancers. These inhibitors can significantly attenuate tumor burden by limiting tumor growth and metastasis. These compounds can also induce DNA damage, cell cycle arrest, apoptosis, and autophagy to promote cancer cell death. Several studies have shown that NAT1 is upregulated in cancer cells. The results of the present study show that the acetylation status of NAT1 is an important factor that might have a relevant role in the progression of cancer.

Acetyl coenzyme A kinetic studies on N-acetylation of environmental carcinogens by human N-acetyltransferase 1 and its NAT1*14B variant.

N-acetyltransferase 1 (NAT1) is a xenobiotic metabolizing enzyme that uses acetyl coenzyme A (AcCoA) as a cofactor for N-acetylation of many carcinogens including aromatic amines and alkylanilines. NAT1 is characterized by single nucleotide polymorphisms (SNPs) that may modulate affinity towards AcCoA. In the current study, we used Chinese hamster ovary (CHO) cells stably transfected with human NAT1*4 (reference allele) or NAT1*14B (variant allele) to measure AcCoA kinetic parameters for N-acetyltransferase activity measurements towards p-aminobenzoic acid (PABA), 4-aminobiphenyl (4-ABP), ß-naphthylamine (BNA), benzidine and 3,4-dimethylaniline (3,4-DMA). Our results showed higher N-acetylation rates for each substrate catalyzed by NAT1*4 compared to NAT1*14B. NAT1*4 exhibited higher affinity to AcCoA when catalyzing the N-acetylation of BNA and benzidine compared to NAT1*14B. The results of the current study provide further insights into differences in carcinogen metabolism among individuals possessing the NAT1*14B haplotype.

560G>A (rs4986782) (R187Q) Single Nucleotide Polymorphism in Arylamine N-Acetyltransferase 1 Increases Affinity for the Aromatic Amine Carcinogens 4-Aminobiphenyl and N-Hydroxy-4-Aminobiphenyl: Implications for Cancer Risk Assessment.

Human arylamine N-acetyltransferase 1 (NAT1) catalyzes the N-acetylation of arylamine carcinogens such as 4-aminobiphenyl (ABP), and following N-hydroxylation, the O-acetylation of N-hydroxy-arylamine carcinogens such as N-hydroxy-ABP (N-OH-ABP). Genetic polymorphisms in NAT1 are linked to cancer susceptibility following exposures. The effects of individual single nucleotide polymorphisms (SNPs) in the NAT1 coding exon on Michaelis-Menten kinetic constants was assessed for ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activity following transfection of human NAT1 into COS-1 cells (SV40-transformed African green monkey kidney cells). NAT1 coding region SNPs 97C > T (rs56318881) (R33stop), 190C > T (rs56379106) (R64W), 559C > T (rs5030839) (R187stop) and 752A > T (rs56172717) (D251V) reduced ABP N- acetyltransferase and N-OH-ABP O-acetyltransferase activity below detection. 21T > G (rs4986992) (synonymous), 402T > C (rs146727732) (synonymous), 445G > A (rs4987076) (V149I), 613A > G (rs72554609) (M205V) and 640T > G (rs4986783) (S241A) did not significantly affect Vmax for ABP N-acetyltransferase or N-OH-ABP O-acetyltransferase. 781G > A (rs72554610) (E261K), and 787A > G (rs72554611) (I263V) slightly reduced ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activities whereas 560G > A (rs4986782) (R187Q) substantially and significantly reduced them. 560G > A (rs4986782) (R187Q) significantly reduced the apparent Km for ABP and N-OH-ABP a finding that was not observed with any of the other NAT1 SNPs tested. These findings suggest that the role of the 560G > A (rs4986782) (R187Q) SNP cancer risk assessment may be modified by exposure level to aromatic amine carcinogens such as ABP.

Proteomic analysis of arylamine N-acetyltransferase 1 knockout breast cancer cells: Implications in immune evasion and mitochondrial biogenesis.

Previous studies have shown that inhibition or depletion of N-acetyltransferase 1 (NAT1) in breast cancer cell lines leads to growth retardation both in vitro and in vivo, suggesting that NAT1 contributes to rapid growth of breast cancer cells. To understand molecular and cellular processes that NAT1 contributes to and generate novel hypotheses in regard to NAT1's role in breast cancer, we performed an unbiased analysis of proteomes of parental MDA-MB-231 breast cancer cells and two separate NAT1 knockout (KO) cell lines. Among 4890 proteins identified, 737 proteins were found significantly (p < 0.01) upregulated, and 651 proteins were significantly (p < 0.01) downregulated in both NAT1 KO cell lines. We performed enrichment analyses to identify Gene Ontology biological processes, molecular functions, and cellular components that were enriched in each data set. Among the proteins upregulated in NAT1 KO cells, pathways associated with MHC (major histocompatibility complex) I-mediated antigen presentation were significantly enriched. This raises an interesting and new hypothesis that upregulation of NAT1 in breast cancer cells may aid them evade immune detection. Multiple pathways involved in mitochondrial functions were collectively downregulated in NAT1 KO cells, including multiple subunits of mitochondrial ATP synthase (Complex V of the electron transport chain). This was accompanied by a reduction in cell cycle-associated proteins and an increase in pro-apoptotic pathways in NAT1 KO cells, consistent with reported observations that NAT1 KO cells exhibit a slower growth rate both in vitro and in vivo. Thus, mitochondrial dysfunction in NAT1 KO cells likely contributes to growth retardation.

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

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.