Arylamine N-acetyltransferase 1 deficiency inhibits drug-induced cell death in breast cancer cells: switch from cytochrome C-dependent apoptosis to necroptosis.

PURPOSE: Arylamine N-acetyltransferase 1 (NAT1) deficiency has been associated with drug resistance and poor outcomes in breast cancer patients. The current study aimed to investigate drug resistance in vitro using normal breast cancer cell lines and NAT1-deficient cell lines to understand the changes induced by the lack of NAT1 that resulted in poor drug response. METHODS: The response to seven chemotherapeutic agents was quantified following NAT1 deletion using CRISPR-Cas 9 in MDA-MB-231 and T-47D cells. Apoptosis was monitored by annexin V staining and caspase 3/7 activity. Cytochrome C release and caspase 8 and 9 activities were measured by Western blots. Caspase 8 was inhibited using Z-IETD-FMK and necroptosis was inhibited using necrostatin and necrosulfonamide. RESULTS: Compared to parental cells, NAT1 depleted cells were resistant to drug treatment. This could be reversed following NAT1 rescue of the NAT1 deleted cells. Release of cytochrome C in response to treatment was decreased in the NAT1 depleted cells, suggesting suppression of the intrinsic apoptotic pathway. In addition, NAT1 knockout resulted in a decrease in caspase 8 activation. Treatment with necrosulfonamide showed that NAT1 deficient cells switched from intrinsic apoptosis to necroptosis when treated with the anti-cancer drug cisplatin. CONCLUSIONS: NAT1 deficiency can switch cell death from apoptosis to necroptosis resulting in decreased response to cytotoxic drugs. The absence of NAT1 in patient tumours may be a useful biomarker for selecting alternative treatments in a subset of breast cancer patients.

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.

NAT1 is a critical prognostic biomarker and inhibits proliferation of colorectal cancer through modulation of PI3K/Akt/mTOR.

The aim of this study was to analyze the correlations between NAT1 and clinicopathological features of and prognosis in colorectal cancer (CRC). RNA sequencing data and clinical information were retrieved from The Cancer Genome Atlas database. Wilcoxon test, logistic regression and Kaplan-Meier method were used to estimate the association between NAT1 and prognosis in CRC. In vitro experiments were conducted to confirm the role of NAT1. NAT1 is significantly less expressed in CRC and independently associated with poor prognosis in CRC patients. The authors further confirmed that expression of NAT1 was significantly lower in SW116 colon cancer cells than in NCM460 cells. Overexpressed NAT1 obviously inhibited the growth of CRC cells by downregulating phosphorylation of the PI3K/Akt/mTOR signaling pathway. NAT1 may be a potential therapeutic target for CRC.

Lay abstract Colorectal cancer (CRC) is a common malignancy worldwide. Because of the limited understanding of the pathogenesis and prognostic factors associated with CRC, the treatment effect in CRC remains poor. In the present study, the authors demonstrate that NAT1 is significantly less expressed in CRC and independently associated with poor prognosis in CRC patients. NAT1 may exert antitumor activity by inhibiting phosphorylation of the PI3K/Akt/mTOR signaling pathway. These results suggest that NAT1 may be a prognostic factor in and therapeutic target for CRC.

NAT1 genetic variation increases asthma risk in children with secondhand smoke exposure.

OBJECTIVE: We previously reported that children exposed to secondhand smoke (SHS) that carried variants in the NAT1 gene had over two-fold higher hair cotinine levels. Our objective was to determine if NAT1 polymorphisms confer increased risk for developing asthma in children exposed to SHS. METHODS: White participants in the Cincinnati Childhood Allergy and Air Pollution Study (n = 359) were genotyped for 10 NAT1 variants. Smoke exposure was defined by hair cotinine and parental report. Asthma was objectively assessed by spirometry and methacholine challenge. Findings were replicated in the Genomic Control Cohort (n = 638). RESULTS: Significant associations between 5 NAT1 variants and asthma were observed in the CCAAPS exposed group compared to none in the unexposed group. There was a significant interaction between NAT1 rs13253389 and rs4921581 with smoke exposure (p = 0.02, p = 0.01) and hair cotinine level (p = 0.048, p = 0.042). Children wildtype for rs4921581 had increasing asthma risk with increasing hair cotinine level, whereas those carrying the NAT1 minor allele had an increased risk of asthma regardless of cotinine level. In the GCC, 13 NAT1 variants were associated with asthma in the smoke-exposed group, compared to 0 in the unexposed group, demonstrating gene-level replication. CONCLUSIONS: Variation in the NAT1 gene modifies asthma risk in children exposed to secondhand-smoke. To our knowledge, this is the first report of a gene-environment interaction between NAT1 variants, smoke exposure, cotinine levels, and pediatric asthma. NAT1 genotype may have clinical utility as a biomarker of increased asthma risk in children exposed to smoke.

Slow N-acetylation as a possible contributor to bladder carcinogenesis.

Bladder cancer (BCa) is an exophytic tumor that presents as either noninvasive confined to the mucosa (NMIBC) or invading the detrusor muscle (MIBC), and was recently further subgrouped into molecular subtypes. Arylamines, major BCa environmental and occupational risk factors, are mainly metabolized by the genetically polymorphic N-acetyltransferases 1, NAT1 and NAT2. In this study, we investigated the association between N-acetyltransferases genetic polymorphism and key MIBC and NMIBC tumor biomarkers and subtypes. A cohort of 250 males with histologically confirmed urothelial BCa was identified. Tumors were genotyped for NAT1 and NAT2 using real-time polymerase chain reaction (PCR), and characterized for mutations in TP53, RB1, and FGFR3 by PCR-restriction fragment length polymorphism. Pathology data and patients' smoking status were obtained from medical records. Pearson χ2 and Fisher exact tests were used to check for associations and interactions. Results show that NAT1 G560 A polymorphism is significantly associated with higher muscle-invasiveness (MIBC vs NMIBC; P = .001), higher tumor grade (high grade vs low grade; P = .011), and higher FGFR3 mutation frequency within the MIBC subgroup (P = .042; .027). NAT2 G857 A polymorphism is also found to be significantly associated with higher muscle-invasiveness (MIBC vs NMIBC; P = .041). Our results indicate that slow N-acetylation is a contributor to bladder carcinogenesis and muscle-invasiveness. These findings highlight NAT1 as a biomarker candidate in BCa and a potential target for drug development.

Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells.

Novel APOBEC1 target 1 (Nat1) (also known as "p97," "Dap5," and "Eif4g2") is a ubiquitously expressed cytoplasmic protein that is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (Eif4g1). We previously showed that Nat1-null mouse embryonic stem cells (mES cells) are resistant to differentiation. In the current study, we found that NAT1 and eIF4G1 share many binding proteins, such as the eukaryotic translation initiation factors eIF3 and eIF4A and ribosomal proteins. However, NAT1 did not bind to eIF4E or poly(A)-binding proteins, which are critical for cap-dependent translation initiation. In contrast, compared with eIF4G1, NAT1 preferentially interacted with eIF2, fragile X mental retardation proteins (FMR), and related proteins and especially with members of the proline-rich and coiled-coil-containing protein 2 (PRRC2) family. We also found that Nat1-null mES cells possess a transcriptional profile similar, although not identical, to the ground state, which is established in wild-type mES cells when treated with inhibitors of the ERK and glycogen synthase kinase 3 (GSK3) signaling pathways. In Nat1-null mES cells, the ERK pathway is suppressed even without inhibitors. Ribosome profiling revealed that translation of mitogen-activated protein kinase kinase kinase 3 (Map3k3) and son of sevenless homolog 1 (Sos1) is suppressed in the absence of Nat1 Forced expression of Map3k3 induced differentiation of Nat1-null mES cells. These data collectively show that Nat1 is involved in the translation of proteins that are required for cell differentiation.

NAT1 genotypic and phenotypic contribution to urinary bladder cancer risk: a systematic review and meta-analysis.

N-acetyltransferase 1 (NAT1), a polymorphic Phase II enzyme, plays an essential role in metabolizing heterocyclic and aromatic amines, which are implicated in urinary bladder cancer (BCa). This systematic review investigates a possible association between the different NAT1 genetic polymorphisms and BCa risk. Medline, PubMed, EMBASE, Scopus, Web of Science, OpenGrey, and BASE databases were searched to identify eligible studies. The random-effect model was used to calculate pooled effects estimates. Statistical heterogeneity was tested with Chi-square and I2. Twenty case-control studies, including 5606 cases and 6620 controls, met the inclusion criteria. Pooled odds ratios (OR) analyses showed a statistically significant difference in NAT1*10 versus non-NAT1*10 acetylators in the total sample (OR: 0.87; 95% CI: 0.79-0.96) but was borderline among Caucasians (OR: 0.88 with 95% CI: 0.77-1.01). No statistically significant differences in BCa risk were found for: NAT1*10 versus NAT1*4 wild type (OR: 0.97; 95% CI: 0.78-1.19), NAT1 'Fast' versus 'Normal' acetylators (OR: 1.03; 95% CI: 0.84-1.27), and NAT1 'Slow' versus 'Fast' (OR: 2.32; 95% CI: 0.93-5.84) or 'Slow' versus 'Normal' acetylators (OR: 1.84; 95% CI: 0.92-3.68). When stratifying by smoking status, no statistically significant differences in BCa risk were found for NAT1*10 versus non-NAT1*10 acetylators among the different subgroups. Our study suggests a modest protective role for NAT1*10 and a possible risk contributory role for slow acetylation genotypes in BCa risk. Further research is recommended to confirm these associations.

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.

Whole-exome sequencing and targeted gene sequencing provide insights into the role of PALB2 as a male breast cancer susceptibility gene.

BACKGROUND: Male breast cancer (MBC) is a rare disease whose etiology appears to be largely associated with genetic factors. BRCA1 and BRCA2 mutations account for about 10% of all MBC cases. Thus, a fraction of MBC cases are expected to be due to genetic factors not yet identified. To further explain the genetic susceptibility for MBC, whole-exome sequencing (WES) and targeted gene sequencing were applied to high-risk, BRCA1/2 mutation-negative MBC cases. METHODS: Germ-line DNA of 1 male and 2 female BRCA1/2 mutation-negative breast cancer (BC) cases from a pedigree showing a first-degree family history of MBC was analyzed with WES. Targeted gene sequencing for the validation of WES results was performed for 48 high-risk, BRCA1/2 mutation-negative MBC cases from an Italian multicenter study of MBC. A case-control series of 433 BRCA1/2 mutation-negative MBC and female breast cancer (FBC) cases and 849 male and female controls was included in the study. RESULTS: WES in the family identified the partner and localizer of BRCA2 (PALB2) c.419delA truncating mutation carried by the proband, her father, and her paternal uncle (all affected with BC) and the N-acetyltransferase 1 (NAT1) c.97C>T nonsense mutation carried by the proband's maternal aunt. Targeted PALB2 sequencing detected the c.1984A>T nonsense mutation in 1 of the 48 BRCA1/2 mutation-negative MBC cases. NAT1 c.97C>T was not found in the case-control series. CONCLUSIONS: These results add strength to the evidence showing that PALB2 is involved in BC risk for both sexes and indicate that consideration should be given to clinical testing of PALB2 for BRCA1/2 mutation-negative families with multiple MBC and FBC cases. Cancer 2017;123:210-218. © 2016 American Cancer Society.

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.

PCR-mediated gene modification strategy for construction of fluorescent protein fusions in Candida parapsilosis.

Candida parapsilosis is a common cause of invasive candidiasis, especially in premature infants, even surpassing Candida albicans as the most frequently identified Candida species in some newborn intensive care units. Whereas many molecular tools are available to facilitate the study of C. albicans, relatively few have been developed for C. parapsilosis. In this study, we show that plasmids harbouring green, yellow and mCherry fluorescent protein sequences, previously developed for expression in C. albicans, can be used to construct fluorescent fusion proteins in C. parapsilosis by PCR-mediated gene modification. Further, the strategy can be used in clinical isolates of C. parapsilosis, which are typically prototrophic, because the plasmids include NAT1, a dominant selectable trait that confers resistance to the antibiotic nourseothricin. Overall, these tools will be useful to yeast researchers who require the ability to visualize C. parapsilosis directly, e.g. in in vitro and in vivo infection models. In addition, this strategy can be used to generate fluorescence in other C. parapsilosis clinical isolates and to tag sequences of interest for protein localization studies. Lastly, the ability to express up to three different fluorescent proteins will allow researchers to visualize and differentiate C. parapsilosis and/or C. albicans clinical isolates from each other in mixed infection models.

Downregulation of NAT1 Expression is Associated with Poor Prognosis and Immune Infiltration in COAD.

Background: An increasing corpus of evidence has identified the involvement of N-acetyltransferase 1 (NAT1), a member of the NAT family, in the progression of various cancers. However, the specific function of NAT1 in colon cancer (COAD) remains elusive. This study aims to decip her the role of NAT1 in COAD and its associated mechanisms. Methods: The Tumor Immunity Evaluation Resource (TIMER), The Cancer Genome Atlas (TCGA), and the Gene Expression Omnibus (GEO) databases were employed to assess the NAT1 expression level in COAD. The differential expression between COAD and normal colon tissue was further validated using quantitative real-time reverse-transcription PCR (RT-qPCR) and Western blot (WB) analyses. Additionally, survival analysis of NAT1 in COAD was carried out using the PrognoScan database and TCGA dataset. The functions of NAT1 were explored through gene set enrichment analysis (GSEA) and immuno-infiltration analysis. Results: There was a significant reduction in NAT1 expression in COAD samples compared to normal tissue. Notably, low NAT1 expression in COAD correlated significantly with various clinical parameters such as tumor stage (T stage, N stage, M stage, pathologic stage), primary therapy outcome, carcinoembryonic antigen (CEA) level, and lymphatic invasion. The downregulation of NAT1 was also strongly linked with poor outcomes in overall survival (OS), progression-free interval (PFI), and disease-specific survival (DSS). Cox regression analysis highlighted NAT1 as an independent prognostic indicator for overall survival in COAD patients. GSEA results revealed NAT1's involvement in multiple pathways, including the neuroactive ligand-receptor interaction, olfactory transduction, olfactory signaling, extracellular matrix receptor interaction, calcium signaling, and focal adhesion pathways. Furthermore, NAT1 expression was found to significantly correlate with infiltration levels of various immune cells. Conclusion: The findings reveal NAT1's potential as a valuable prognostic biomarker for COAD. Moreover, its associated mechanisms offer insights that might pave the way for therapeutic interventions for COAD patients.

NAT1 inhibits liver metastasis of colorectal cancer by regulating EMT and glycolysis.

Metastasis is the primary cause of cancer-related deaths, and colorectal cancer (CRC) liver metastasis is a major poor prognostic factor in CRC. NAT1 (N-acetyltransferase 1) plays a crucial role in the invasive and metastatic processes of colorectal cancer. The role and molecular mechanism of NAT1 on tumor cells were verified by establishing a cell model of overexpression and knockdown of NAT1, and further verified by establishing a liver metastasis model of colorectal cancer for animal experiments. In vivo and in vitro experiments have demonstrated that overexpression of NAT1 reduces the ability of metastasis and invasion of colorectal cancer cells. NAT1 overexpression inhibits the PI3K/AKT/mTOR signaling pathway, thereby suppressing the EMT (epithelial-mesenchymal transition) process and glycolytic ability of tumor cells. Additionally, decreased glycolytic ability results in reduced VEGF (Vascular endothelial growth factor) expression in colorectal cancer cells. The decreased VEGF expression leads to decreased angiogenesis and vascular permeability in liver metastases, ultimately reducing the occurrence of liver metastasis. Our findings highlight that overexpression of NAT1 significantly inhibits the PI3K/AKT/mTOR signaling pathway, thereby suppressing EMT, glycolytic ability, and VEGF expression in colorectal cancer cells, collectively preventing the development of liver metastasis.

Association of the cytochrome P450 and arylamine N-acetyltransferase gene polymorphisms with the incidence of head and neck cancer in Polish population.

OBJECTIVES: Head and neck cancer (HNC) is one of the most common cancers. Most exogenous HNC is head and neck squamous cell carcinomas. Scientists are striving to develop diagnostic tests that will allow the prognosis of HNC. The aim of the study was to determine the risk of HNC. The research concerned changes caused by polymorphisms in genes encoding proteins responsible for the metabolism of xenobiotics. MATERIAL AND METHODS: In group of 280 patients with HNC, the occurrence of polymorphic variants in NAT1(rs72554606), NAT2(rs1799930), CYP1A(rs1799814), CYP2D(rs3892097) were studied with TaqMan technique. The control group consisted of 260 cancer free people. The TNM scale was analyzed. Gene interactions of genotyped polymorphisms were investigated. The effects of smoking and alcohol consumption on HNC were assessed. RESULTS: The results indicated an increased risk of HNC in NAT1 polymorphisms in the GC genotype (OR = 1.772, 95% CI: 1.184-2.651, p = 0.005) and NAT2 polymorphism in the GA genotype (OR = 1.506, 95% CI: 1.023-2.216, p = 0.037). The protective phenomenon in the CYP1A polymorphism the GT genotype (OR = 0.587, 95% CI: 0.381-0.903, p = 0.015) and the TT genotype (OR = 0.268, 95% CI: 0.159-0.452, p = 0.001). The coexistence of GA-GC polymorphisms (OR = 2.687, 95% CI: 1.387-5.205, p = 0.003) in NAT2-NAT1 genes increases the risk of HNC. Risk-reducing effect in the polymorphism GG-GT (OR = 0.340, 95% CI: 0.149-0.800, p = 0.011), GG-TT (OR = 0.077, 95% CI: 0.028-0.215, p < 0.0001), GA-TT (OR = 0.250, 95% CI: 0.100-0.622, p = 0.002), AA-GT (OR = 0.276, 95% CI: 0.112-0.676, p = 0.002) in NAT2-CYP1A genes. In the CYP2D-CYP1A genes in the polymorphisms CT-CC (OR = 0.338, 95% CI: 0.132-0.870, p = 0.020), TT-GG (OR = 0.100, 95% CI: 0.027-0.359, p = 0.001), TT-GC (OR = 0.190, 95% CI: 0.072-0.502, p = 0.0004), TT-CC (OR = 0.305, 95% CI: 0.107-0.868, p = 0.024). Correlation was noted between cigarette smoking and HNC (OR = 7.297, 95% CI: 4.989-10.674, p < 0.0001) and consuming alcohol (OR = 1.572, 95% CI: 1.003-2.464, p = 0.047). CONCLUSIONS: The CYP1A polymorphism shows a protective association with HNC. On the other hand, NAT2, NAT1 polymorphism influence the susceptibility to developing HNC. The coexistence of the NAT2-NAT1 genotypes increases the risk of HNC. In contrast, NAT1-CYP1A and CYP1A-CYP2D reduce this risk. Smoking and alcohol consumption increase the incidence of HNC. Int J Occup Med Environ Health. 2023;36(6):812-24.

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.

Weighted Gene Co-expression Network Analysis Identified a Novel Thirteen-Gene Signature Associated With Progression, Prognosis, and Immune Microenvironment of Colon Adenocarcinoma Patients.

Colon adenocarcinoma (COAD) is one of the most common malignant tumors and has high migration and invasion capacity. In this study, we attempted to establish a multigene signature for predicting the prognosis of COAD patients. Weighted gene co-expression network analysis and differential gene expression analysis methods were first applied to identify differentially co-expressed genes between COAD tissues and normal tissues from the Cancer Genome Atlas (TCGA)-COAD dataset and GSE39582 dataset, and a total of 309 overlapping genes were screened out. Then, our study employed TCGA-COAD cohort as the training dataset and an independent cohort by merging the GES39582 and GSE17536 datasets as the testing dataset. After univariate and multivariate Cox regression analyses were performed for these overlapping genes and overall survival (OS) of COAD patients in the training dataset, a 13-gene signature was constructed to divide COAD patients into high- and low-risk subgroups with significantly different OS. The testing dataset exhibited the same results utilizing the same predictive signature. The area under the curve of receiver operating characteristic analysis for predicting OS in the training and testing datasets were 0.789 and 0.868, respectively, which revealed the enhanced predictive power of the signature. Multivariate Cox regression analysis further suggested that the 13-gene signature could independently predict OS. Among the 13 prognostic genes, NAT1 and NAT2 were downregulated with deep deletions in tumor tissues in multiple COAD cohorts and exhibited significant correlations with poorer OS based on the GEPIA database. Notably, NAT1 and NAT2 expression levels were positively correlated with infiltrating levels of CD8+ T cells and dendritic cells, exhibiting a foundation for further research investigating the antitumor immune roles played by NAT1 and NAT2 in COAD. Taken together, the results of our study showed that the 13-gene signature could efficiently predict OS and that NAT1 and NAT2 could function as biomarkers for prognosis and the immune response in COAD.

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.

MicroRNA-6744-5p promotes anoikis in breast cancer and directly targets NAT1 enzyme.

Objective: Anoikis is apoptosis that is induced when cells detach from the extracellular matrix and neighboring cells. As anoikis serves as a regulatory barrier, cancer cells often acquire resistance towards anoikis during tumorigenesis to become metastatic. MicroRNAs (miRNAs) are short strand RNA molecules that regulate genes post-transcriptionally by binding to mRNAs and reducing the expression of its target genes. This study aimed to elucidate the role of a novel miRNA, miR-6744-5p, in regulating anoikis in breast cancer and identify its target gene. Methods: An anoikis resistant variant of the luminal A type breast cancer MCF-7 cell line (MCF-7-AR) was generated by selecting and amplifying surviving cells after repeated exposure to growth in suspension. MiRNA microarray analysis identified a list of dysregulated miRNAs from which miR-6744-5p was chosen for overexpression and knockdown studies in MCF-7. Additionally, the miRNA was also overexpressed in a triple-negative breast cancer cell line, MDA-MB-231, to evaluate its ability to impair the metastatic potential of breast cancer cells. Results: This study showed that overexpression and knockdown of miR-6744-5p in MCF-7 increased and decreased anoikis sensitivity, respectively. Similarly, overexpression of miR-6744-5p in MDA-MB-231 increased anoikis and also decreased tumor cell invasion in vitro and in vivo. Furthermore, NAT1 enzyme was identified and validated as the direct target of miR-6744-5p. Conclusions: This study has proven the ability of miR-6744-5p to increase anoikis sensitivity in both luminal A and triple negative breast cancer cell lines, highlighting its therapeutic potential in treating breast cancer.

Genetic variants of N-acetyltransferases 1 and 2 (NAT1 and NAT2) in cynomolgus and rhesus macaques.

In humans, polymorphic N-acetyltransferases NAT1 and NAT2 are important enzymes that metabolize endogenous and exogenous compounds, including drugs. These enzymes exhibit considerable inter-individual variability in humans. The cynomolgus macaque is a nonhuman primate species that is widely used in drug metabolism studies. NAT1/2 in these macaques have molecular and enzymatic similarities to their human orthologs; however, genetic polymorphisms in NAT1/2 have not been fully investigated in this species. In this study, the resequencing of NAT1 and NAT2 in 114 cynomolgus macaques and 19 rhesus macaques found 15 non-synonymous variants for NAT1 and 11 non-synonymous variants and 1 insertion/deletion variant for NAT2. Nine (60%) and five (33%) NAT1 variants and seven (67%) and three (25%) NAT2 variants were unique to cynomolgus and rhesus macaques, respectively. Functional characterization of the mutant enzymes was carried out using cynomolgus NAT1 and NAT2 proteins heterologously expressed in Escherichia coli. Compared with wild-type NAT1, the D122N NAT1 variant showed substantially lower acetylation activities toward p-aminobenzoic acid but had higher acetylation activities toward isoniazid. Moreover, liver cytosolic fractions from cynomolgus macaques homozygous for T98A NAT2 showed significantly lower acetylation activities toward isoniazid than wild-type NAT2; similar results were obtained for recombinant T98A NAT2. Interestingly, all the rhesus macaques analyzed were homozygous for T98A. These findings indicate that polymorphic NAT1/2 variants in cynomolgus and rhesus macaques, especially the T98A NAT2 variant, could account for the inter-animal and/or inter-lineage variabilities of NAT-dependent drug metabolism in macaques.