Neville J. Butcher

Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that also has a role in cancer cell growth and metabolism. Recently, it was reported that NAT1 undergoes lysine acetylation, an important post-translational modification that can regulate protein function. In the current study, we use site-directed mutagenesis to identify K100 and K188 as major sites of lysine acetylation in the NAT1 protein. Acetylation of ectopically expressed NAT1 in HeLa cells was decreased by C646, an inhibitor of the protein acetyltransferases p300/CBP. Recombinant p300 directly acetylated NAT1 in vitro. Acetylation of NAT1 was enhanced by the sirtuin inhibitor nicotinamide but not by the histone deacetylase inhibitor trichostatin A. Co-transfection of cells with NAT1 and either SIRT 1 or 2, but not SIRT3, significantly decreased NAT1 acetylation. NAT1 activity was evaluated in cells following nicotinamide treatment to enhance acetylation or co-transfection with SIRT1 to inhibit acetylation. The results indicated that NAT1 acetylation altered its enzyme kinetics, suggesting changes in acetyl coenzyme A binding. In addition, acetylation attenuated the allosteric effects of ATP on NAT1. Taken together, this study shows that NAT1 is acetylated by p300/CBP in situ and is deacetylated by the sirtuins SIRT1 and 2. It is hypothesized that post-translational modification of NAT1 by acetylation at K100 and K188 may modulate NAT1 effects in cells. SIGNIFICANCE STATEMENT There is growing evidence that arylamine N-acetyltransferase 1 has an important cellular role, in addition to xenobiotic metabolism. Here, we show that NAT1 is acetylated at K100 and K188 and changes in protein acetylation equilibrium can modulate its activity in cells.

SULT4A1 is a brain-selective sulfotransferase-like protein that has recently been shown to be essential for normal neuronal development in mice. In the present study, SULT4A1 was found to co-localize with SULT1A1/3 in human brain neurons. Using immunoprecipitation, SULT4A1 was shown to interact with both SULT1A1 and 1A3 when expressed in human cells. Mutation of the conserved dimerization motif located in the C-terminus of the sulfotransferases prevented this interaction. Both ectopically expressed and endogenous SULT4A1 decreased SULT1A1/3 protein levels in neuronal cells, which was also prevented by mutation of the dimerization motif. During differentiation of neuronal SH-SY5Y cells, there was a loss in SULT1A1/3 protein but an increase in SULT4A1 protein. This resulted in an increase in the toxicity of dopamine, a substrate for SULT1A3. Inhibition of SULT4A1 using siRNA abrogated the loss in SULT1A1/3 and reversed dopamine toxicity. These results show a reciprocal relationship between SULT4A1 and the other sulfotransferases suggesting that it may act as a chaperone to control the expression of SULT1A1/3 in neuronal cells. SIGNIFICANCE STATEMENT The catalytically inactive sulfotransferase SULT4A1 may regulate the function of other SULTs by interacting with them via a conserved dimerization motif. In neuron-like cells, SULT4A1 is able to modulate dopamine toxicity by interacting with SULT1A3, potentially decreasing the metabolism of dopamine.

Reducted arylamine N-acetyltransferase (NAT1) in breast cancers is associated with poor patient survival. NAT1 has also been associated with changes in cancer cell survival and invasion both in vitro and in vivo. Here, we report the effects of NAT1 in cancer cell invasion by addressing its role in adherence, migration, and invasion in vitro. The NAT1 gene was deleted in MDA-MB-231, HT-29 and HeLa cells using CRISPR/Cas9 gene editing. Loss of NAT1 increased adherence to collagen in all three cell-lines but migration was unaffected. NAT1 deletion decreased invasion and induced changes to cell morphology. These effects were independent of matrix metalloproteinases but were related to integrin ITGαV expression. The data suggest NAT1 is important in adhesion and invasion through integrin expression.

Arylamine N-acetyltransferase 1 (NAT1) is a drug metabolizing enzyme that significantly influences cancer cell proliferation and survival. However, the mechanism for these effects is unknown. Because of previous observations that NAT1 inhibition significantly decreases invasiveness, we investigated the expression of the metalloproteinase MMP9 in human breast cancer samples and in cancer cells. We found a negative correlation between the expression of NAT1 and MMP9 in 1904 breast cancer samples. Moreover, when NAT1 was deleted in highly invasive breast cancer cells, MMP9 mRNA and protein significantly increased, both of which were reversed by re-introducing NAT1 into the knockout cells. Following NAT1 deletion, there was an increased association of acetylated histone H3 with the SMYD3 element in the MMP9 promoter, consistent with an increase in MMP9 transcription. NAT1 deletion also up-regulated Hif1-α. Treatment of the NAT1 knockout cells with siRNA directed towards HIF1-α mRNA inhibited the increased expression of MMP9. Taken together, these results show a direct inverse relationship between NAT1 and MMP9 and suggest that HIF1-α may be essential for the regulation of MMP9 expression by NAT1. SIGNIFICANCE STATEMENT The expression of the enzyme arylamine N-acetyltransferase 1 was found to be negatively correlated with MMP9 expression in tumor tissue from breast cancer patients. In cells, arylamine N-acetyltransferase 1 regulated MMP9 expression at a transcriptional level. This finding is highly significant as it may explain some of the pathological features associated with changes in arylamine N-acetyltransferase 1 expression in cancer.

Sulfotransferase 4A1 (SULT4A1) is a sulfotransferase-like protein that is highly conserved between species. In human tissues, there are 2 transcripts, one that produces a full length protein and one that produces an unstable truncated protein. The second transcript, which includes a pseudo-exon between exons 6 and 7 (6p), is widely expressed while the first is more restricted. Differentiation of neuronal cells results in the removal of the pseudo-exon and subsequent SULT4A1 protein expression. Recent studies with SULT4A1 knockout mice showed that the protein is essential for normal development and that its absence leads to a severe neurological phenotype. Here, the regulation of SULT4A1 6p splicing was investigated during neuronal differentiation using SH-SY5Y cells, human induced pluripotent stem cells and mouse embryonic tissue. In all 3 models, pseudo-exon 6p was removed during differentiation, resulting in stable SULT4A1 protein expression. Using a minigene splicing assay, a region upstream of pseudo-exon 6p was identified that is essential for correct splicing of SULT4A1 mRNA. Within this region, there were binding motifs for 4 RNA processing factors (MBNL-1, MBNL-2, CELF-1 and CELF-2). Time-dependent changes in SULT4A1 protein and MBNL/CELF protein during differentiation supported their role in correctly splicing the SULT4A1 mRNA. Furthermore, ectopic expression of each factor produced efficient splicing in the minigene assay as well as correct splicing of the endogenous SULT4A1 mRNA. These results show that SULT4A1 mRNA is a target for MBNL/CELF-dependent splicing, which may be essential in producing stable, function SULT4A1.

Arylamine N-acetyltransferase 1 (NAT1) is a drug metabolizing enzyme that has been associated with cancer cell proliferation in vitro and with survival in vivo. NAT1 expression has been associated with the estrogen receptor and it has been proposed as a prognostic marker for estrogen receptor positive cancers. However, little is known about the distribution of NAT1 mRNA across an entire patient population or its effects on outcomes. To address this, gene expression data from breast cancer patient cohorts were investigated to identify sub-populations based on the level of NAT1 expression. Patient survival and drug response was examined to determine whether NAT1 mRNA levels influenced any of these parameters. NAT1 expression showed a trimodal distribution in breast cancer samples (n = 1980) but not in tumor tissue from ovarian, prostate, cervical or colorectal cancers. In breast cancer, NAT1 mRNA in each sub-population correlated with a separate set of genes suggesting different mechanisms of NAT1 gene regulation. Kaplan-Meier plots showed significantly better survival in patients with highest NAT1 mRNA compared to those with intermediate or low expression. While NAT1 expression was elevated in estrogen receptor-positive patients, it did not appear to be dependent on estrogen receptor expression. Overall survival was analyzed in patients receiving no treatment, hormone therapy or chemotherapy. NAT1 expression correlated strongly with survival in the first 5 years in those patients receiving chemotherapy but did not influence survival in the other two groups. This suggests that low NAT1 expression is associated with chemo-resistance. The sensitivity of NAT1 mRNA levels as a single parameter to identify non-responders to chemotherapy was 0.58 at a log(2) < 6.5. NAT1 mRNA can be used to segregate breast cancer patients into sub-populations that demonstrate different overall survival. Moreover, low NAT1 expression shows a distinct poor response to chemotherapy. Analysis of NAT1 expression may be useful for identifying specific individuals who would benefit from alternative therapy or drug combinations. However, additional information is required to increase the sensitivity of identifying non-responders.