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Retrospective analysis of estrogen receptor 1 and N-acetyltransferase gene expression in normal breast tissue, primary breast tumors, and established breast cancer cell lines

The expression levels of estrogen receptor 1 (ESR1), arylamine N-acetyltransferase 1 (NAT1), and arylamine N-acetyltransferase 2 (NAT2) are implicated in breast cancer; however, their co-expression profiles in normal breast tissue, primary breast tumors and established breast cancer cell lines are undefined. NAT1 expression is widely reported to be associated with ESR1 expression and is frequently investigated in breast cancer etiology. Furthermore, the NAT2 phenotype has been reported to modify breast cancer risk in molecular epidemiological association studies. Understanding the relationships between the expression levels of these genes is essential to understand their role in breast cancer etiology and treatment. In the present study, NAT1, NAT2 and ESR1 expression data were accessed from repositories of RNA-Seq data covering 57 breast cancer cell lines, 1,043 primary breast tumors and 99 normal breast tissues. The relationships between gene expression, and between NAT1 activity and RNA expression in breast cancer cell lines were evaluated using non-parametric statistical analyses. Differences in gene expression in each dataset, as well as gene expression differences in normal breast tissue compared to primary breast tumors, and stratification by estrogen receptor status were determined. NAT1 and NAT2 mRNA expression were detected in normal and primary breast tumor tissues; NAT1 expression was much higher than NAT2. NAT1 and ESR1 expression were strongly associated, whereas NAT2 and ESR1 expression were not. Although NAT1 and NAT2 expression were associated, the magnitude was moderate. NAT1, NAT2, and ESR1 expression were increased in primary breast tumor tissue compared with normal breast tissue; however, the magnitude and significance of the differences were lower for NAT2. Analysis of NAT1, NAT2, and ESR1 expression in normal and primary breast tissues and breast cancer cell lines suggested that NAT1 and NAT2 expression are regulated by distinctive mechanisms, whereas NAT1 and ESR1 expression may have overlapping regulation. Defining these relationships is important for future investigations into breast cancer prevention.

Although NAT1 and NAT2 catalyze N-acetylation, their roles in breast cancer etiology may differ. Numerous studies have investigated possible roles for NAT1 in breast cancer etiology and progression (18)(19)(20)(21)(22)(23)(24), given the association between increased expression of NAT1 and estrogen receptor (ER)-positive breast cancers (25)(26)(27)(28)(29)(30)(31). Notably, NAT1 expression is not directly regulated by estrogens or dihydrotestosterone (32), thus suggesting that there may be a common regulatory element between NAT1 and ESR1. Furthermore, congenic rats expressing higher NAT2 activity (orthologous to human NAT1) have been reported to exhibit greater carcinogen-induced mammary tumor susceptibility independent of carcinogen metabolism (22). In addition, SNPs in NAT2 have been well described and have been revealed to influence acetylation rates of many known carcinogens; an association between NAT2 genotype with breast cancer risk among smokers has been reported (33). Since NAT1 and NAT2 may have different roles in breast cancer, it is important to analyze relationships between the expression levels of these isozymes.
The mRNA expression levels of NAT1 and NAT2 have been detected by reverse transcription-polymerase chain reaction (RT-PCR) in human mammary tissue (34,35). NAT1 N-acetylation activity has been widely reported in normal breast tissue and breast tumor tissue (34,(36)(37)(38)(39)(40), whereas NAT2 N-acetylation activity has not been observed as consistently; when NAT2 activity is observed the activity is much lower than NAT1 activity (34,38,39). In addition, since NAT1 and NAT2 have overlapping substrate specificities, activity studies of the two isozymes can be complex. For example, Deitz probed human mammary tissue samples for NAT1 and NAT2 activities with p-aminobenzoic acid (PABA; selective for NAT1) and sulfamethazine (SMZ; selective for NAT2), and reported that SMZ was acetylated by NAT1 at very low levels (40). By normalizing the SMZ N-acetylation activity to NAT1 activity, Deitz demonstrated that the SMZ N-acetylation activity was most likely catalyzed by NAT1 rather than NAT2. NAT1 and NAT2 activities have also been reported in rat mammary tissues (41).
Wakefield et al profiled NAT1 expression and activity in seven breast cancer cell lines (MCF-7, T47D, ZR-75-1, Cal51, MDA-MB-231, MDA-MB-437 and MDA-MB-453) and detected NAT1 mRNA expression and activity in all seven cell lines (28); however, NAT2 expression and activity were not investigated. In addition, NAT2 mRNA has been detected in MCF-7 breast cancer cells at very low levels (35); however, NAT1 was not measured at the same time preventing a direct comparison of expression between the two isozymes. Bradshaw et al detected NAT1 and NAT2 by western blotting in the ER-positive breast cancer cell line MCF-7; however, the expression levels were not compared between the two proteins (42).
Limited studies have investigated the expression profiles of these isozymes together in breast tissues. Based on limited data, it has been hypothesized that NAT2 expression is very low in breast tissue and negligible in comparison to NAT1 expression; however, previous investigations have not addressed this hypothesis rigorously or comprehensively. To gain a better understanding of the relationship between NAT1 and NAT2 in breast tissues the present study evaluated the RNA expression levels of each in breast cancer cell lines, breast tumor tissue, and normal breast tissue. In addition, this study evaluated the extent to which established breast cancer cell lines reflect the NAT expression profile observed in primary breast tumors and normal breast tissue. Since NAT1 and NAT2 are so similar in terms of sequence, structure and substrates, and the association between NAT1 and ESR1 has been well established, the present study also evaluated the relationship between NAT2 and ESR1 expression in breast tissues. Since inhibition of NAT1 activity is under investigation for breast cancer prevention and treatment, understanding the relationships between NAT1, NAT2 and ESR1 is of great importance.

Materials and methods
Acquisition of publicly available data from the Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) data repositories. RNA expression (RNA-Seq) data for ESR1, NAT1 and NAT2 in established breast cancer cell lines were accessed on 8/11/17 (n=57) from the CCLE (43) Statistical analyses. Shapiro-Wilk tests were conducted to determine if the expression of the genes under study were approximately normally distributed. Significant evidence of departures from approximate normality was observed; therefore, non-parametric statistical techniques were employed for subsequent analyses. Spearman's correlation was used to evaluate the RNA expression levels between gene pairs (i.e. ESR1 and NAT1, ESR1 and NAT2, NAT1 and NAT2). Differences in the mRNA expression levels of NAT1 and NAT2 in each dataset, and differences in RNA expression between primary breast tumor samples and normal breast tissue samples, were evaluated using the Wilcoxon rank-sum test; median values were compared to determine fold-differences.
RNA expression data for each gene were stratified by ER status (+ or -) as defined in the literature (46)(47)(48) for the CCLE data, or as determined by immunohistochemistry during sample collection and cataloging for TCGA data. Differences in gene expression following stratification were evaluated using Wilcoxon rank-sum tests for each gene; median values were compared to determine fold-differences. A total of 10 of the breast cancer cell lines had either conflicting or unknown ER status in the literature.  (28). The association between previously reported NAT1 activity and NAT1 RNA expression data for the same cell lines in the CCLE repository was evaluated. All statistical analyses were performed in R: A Language and Environment for Statistical Computing, version 3.4.2 (49).

Results
Association between NAT1 and ESR1, NAT2 and ESR1, and NAT1 and NAT2. NAT1 RNA and ESR1 RNA were significantly correlated (P<0.0001 for all) at moderately high magnitudes in breast cancer cell lines (Spearman rho ρ=0.59; Fig. 1A and B), human primary breast tumors (ρ= 0.59; Fig. 1C and D) and normal breast tissue (ρ= 0.57; Fig. 1E and F). A significant (P<0.005 for all) association between ESR1 and NAT2 expression was observed, although the magnitude of the association was low and varied across datasets. The primary breast tumor dataset exhibited the weakest association (ρ= 0.16; Fig. 1C and D), whereas the normal breast tissue (ρ= 0.38; Fig. 1E and F) and breast cancer cell line (ρ= 0.39; Fig. 1A and B) datasets exhibited similar, albeit low, association. Strong evidence of an association (P<0.0001 for all) between NAT1 RNA and NAT2 RNA levels was observed in all three datasets, with moderately high magnitude in the breast cancer cell lines (ρ=0.64; Fig. 1A and B). The primary breast tumor and normal breast tissue datasets exhibited interdependence similar to each other (ρ= 0.43 and 0.46, respectively; Figs. 1C-F); however, the association was lower than that observed in the breast cancer cell lines.
Comparison of NAT1 and NAT2 expression. NAT1 RNA expression in breast cancer cell lines, primary breast tumors, and normal breast tissue was significantly higher compared In TCGA dataset, normal breast tissue samples were collected from patients in which primary breast tumor samples were also collected (but only for 99 individuals), allowing comparison of gene expression between normal breast tissue and primary tumor breast tissue within single individuals. In the primary breast tumor samples, only nine of the 984 samples had higher NAT2 RNA expression than NAT1; of those nine samples, two were ER + and seven were ER -, and only one sample had a corresponding normal breast tissue sample. Notably, in that individual's normal breast tissue sample, NAT2 RNA expression was not higher than NAT1 RNA expression. In the normal breast tissue samples, only one of the 92 samples had higher NAT2 RNA expression than NAT1; the corresponding primary breast tumor sample from the same patient had lower NAT2 than NAT1.
Comparison of gene expression between ER + and ERsamples. ESR1 and NAT1 gene expression were significantly increased, 86-and 2.6-fold, respectively, in ER + breast cancer cell lines (P<0.0001 for both; Fig. 3A), whereas NAT2 gene expression did not significantly vary between ER + and ERbreast cancer cell lines (P>0.05; Fig. 3A). Of the breast cancer cell lines with ER status defined in the literature (46)(47)(48), a connection between ESR1 RNA expression and the reported ER status has been observed. According to the literature, samples in the dataset with ESR1 RNA expression <1.7 RPKM were defined as ER -, whereas samples with ESR1 expression >2.3 RPKM were defined as ER + . The expression levels of all three genes were significantly higher in ER + primary breast tumor samples (P<0.0001 for all; Fig. 3B); however. the fold-change between NAT2 expression in ER + and ERsamples was smaller (1.8-fold difference) than for NAT1 and ESR1. In comparison, ESR1 and NAT1 were ~108-and 27-fold higher, respectively. The expression levels of genes were not significantly different between ER + and ERnormal breast tissue samples (P>0.05 for all; Fig. 3C). Most of the breast cancer cell lines were ER -, whereas most of the primary breast tumor and normal breast tissue samples were ER + .
Comparison of NAT1, NAT2, and ESR1 gene expression between normal breast tissue and primary breast tumors. Differences in gene expression between normal breast tissue and primary breast tumor tissue were evaluated for each gene, ESR1, NAT1, and NAT2. More spread was observed in the primary breast tumor samples compared with the normal breast tissue samples for each gene. ESR1 and NAT1 gene expression were significantly elevated 2.5-and 5.9-fold, respectively, in primary breast tumor samples compared with normal breast tissue samples (P<0.0001 for both; Fig. 4). NAT2 expression was also significantly higher in primary breast tumor samples compared with normal breast tissue samples, but at a lower significance and fold-change (1.4fold) than ESR1 and NAT1 (P<0.05; Fig. 4).
Relationship between previously reported NAT1 N-acetylation activity and NAT1 RNA expression. NAT1 N-acetylation  genes in breast cancer cell lines, primary breast tumor tissue, and normal breast tissue stratified by ER status were evaluated by Wilcoxon rank-sum test; *** P<0.001; NS, not significant. Boxplots are color-coded according to ER status; pink boxplots, ERsamples; blue boxplots, ER + samples. In the boxplots, the solid black line represents the median, the upper hinge represents the 75th quartile and the lower hinge represents the 25th quartile. The upper whisker represents the largest observation less than or equal to the upper hinge + 1.5 x IQR, the lower whisker represents the smallest observation greater than or equal to the lower hinge -1.5 x IQR. (A) ESR1 and NAT1 RNA expression were significantly higher in ER + breast cancer cell lines compared with ERbreast cancer cell lines. NAT2 RNA expression was not significantly different in ER + breast cancer cell lines compared with ERbreast cancer cell lines. A total of 10 cell lines had either conflicting reports or no available data for ER status in the literature and were excluded from the analysis.  Boxplots are color-coded according to tissue type; green boxplots, normal breast tissue samples; blue boxplots, primary breast tumor samples. In the boxplots, the solid black line represents the median, the upper hinge represents the 75th quartile and the lower hinge represents the 25th quartile. The upper whisker represents the largest observation less than or equal to the upper hinge + 1.5 x IQR, the lower whisker represents the smallest observation greater than or equal to the lower hinge -1.5 * IQR. For all genes, more spread was observed in data from the primary breast tumor samples compared with the normal breast tissue samples. ESR1 and NAT1 gene expression were significantly elevated in primary tumor tissue compared with normal breast tissue. NAT2 expression was also significantly higher in primary tumor tissue compared with normal breast tissue, but at a lower significance than ESR1 and NAT1. IQR, interquartile range; ESR1, estrogen receptor 1; NAT1, arylamine N-acetyltransferase 1; NAT2, arylamine N-acetyltransferase 2; RSEM, RNA-Seq by Expectation-Maximization.

Discussion
The present study analyzed established breast cancer cell lines and samples from patients with breast cancer to evaluate the extent to which breast cancer cell lines serve as appropriate models for NAT1, NAT2 and ESR1 expression in breast tumors. Overall, the present findings demonstrated a strong association between NAT1 and ESR1 expression, which is in agreement with previous reports that NAT1 and ESR1 are positively associated (25)(26)(27)(28)(29)(30)(31), and this association was observed in all three sample types at approximately the same magnitude. These findings suggested that breast cancer cell lines may accurately reflect this relationship and provide a useful model for further research into the relationship. It is well known that ESR1 expression is frequently altered in breast cancer; therefore, the decrease in association between NAT2 and ESR1 in primary breast tumors compared with normal breast tissue samples and established breast cancer cell lines may be due to more dysregulation of ESR1 than NAT2 in primary breast tumors. The results of an analysis between NAT2 and ESR1 expression suggested that, while NAT2 and ESR1 are associated, the magnitude is low.
Interdependence between NAT1 and NAT2 expression was moderately high in the breast cancer cell line dataset, but substantially lower in the primary breast tumor and normal breast tissue datasets. Additionally, the strength of the association between NAT1 and NAT2 in the breast cancer cell line dataset was similar to the strength of the association observed between NAT1 and ESR1 in that dataset; however, in the primary breast tumor and normal breast tissue datasets, the association between NAT1 and NAT2 was lower. These findings suggested that breast cancer cell lines may over-represent the interdependence between NAT1 and NAT2, and not fully replicate the relationship observed in primary breast tumors or normal breast tissue.
In the breast cancer cell line data there appears to be a cut-off (between 1.7 and 2.3 RPKM) linking ESR1 RNA expression and the reported ER status of the breast cancer cell lines. This may provide a method to predict the ER status of breast cancer cell lines that currently have conflicting or unknown ER status in the literature. Using that method, it may be predicted that the HCC1500 and HCC1419 cell lines are ER + , whereas the HMC-1-8, Hs 742.T, Hs 343.T, Hs 739.T, HMEL, Hs 274.T, Hs 281.T and Hs 606.T cell lines are ER -. Notably, although 67-82% of breast cancers are ER + (51) and most of the primary breast tumor samples were ER + , the majority of established breast cancer cell lines are ER -.
NAT1 and NAT2 RNA expression were reported in almost all samples included in the present study, which concurs with previously published results that have detected NAT1 and NAT2 mRNA by RT-PCR in human mammary tissue in smaller cohorts (34)(35)(36). NAT1 RNA expression was significantly higher than NAT2 RNA expression in the breast cancer cell lines, primary breast tumor samples and normal breast tissue. In addition, with only a few exceptions, NAT1 RNA expression was always higher than NAT2 RNA expression in matched samples from the breast cancer cell line, primary breast tumor sample and normal breast tissue sample datasets, thus supporting previous findings that indicated NAT1 transcripts were 2-to 3-fold higher than NAT2 transcripts in Figure 6. NAT1 and NAT2 association in breast cancer cell lines. Association between NAT1 and NAT2 RNA expression was analyzed in breast cancer cell lines (each labeled in this figure). Each dot represents a single breast cancer cell line and is color-coded according to ER status; pink dots, ERsamples; blue dots, ER + samples; black dots, samples with unknown or conflicting ER status in the literature (46)(47)(48). NAT1 and NAT2 RNA expression was significantly associated in breast cancer cell lines (P<0.0001, ρ= 0.64).
human mammary tissues (52). The UACC-893 cell line, the only breast cancer cell line observed in this study to express higher NAT2 RNA than NAT1 RNA, is an ERand progesterone receptor-negative cell line that has a ~20-fold amplification of the human epidermal growth factor receptor 2/neu oncogene sequence. Further study of this cell line may aid in the identification of additional regulatory mechanisms of NAT1 and/or NAT2, since it expresses a unique profile of NAT1 and NAT2 compared with the other breast cancer cell lines.
While NAT1 expression was reported in all 57 breast cancer cell lines in the present study, 15 of those breast cancer cell lines had no reported NAT2 RNA expression (Fig. 6). The cell lines with no detected NAT2 RNA are plotted at ~-6.6 log 2 RPKM NAT2. One of those 15 cell lines, MCF-7, has previously been reported to express NAT2 RNA expression (35,53) albeit at very low levels. One reason for the difference in observation between this study and the previous studies may be that the detection threshold for NAT2 was higher when measured by RNA-Seq for the CCLE dataset than in the previous studies. Additionally, in the previous studies that detected NAT2 RNA in the MCF-7 breast cancer cell line, NAT1 RNA was not measured at the same time; therefore, direct comparisons of the isozymes was not possible. To the best of our knowledge, NAT2 RNA expression has not been investigated in any of the other 56 breast cancer cell lines until this study. The results of this study indicated that NAT2 may be expressed in breast tissues and expression should be considered when studying NAT1, due to their overlapping substrate specificities and the high degree of structural similarity.
In normal breast tissue samples no significant difference in gene expression for ESR1, NAT1 and NAT2 was observed when data was stratified by ER status. However, in the primary breast tumor samples and in the breast cancer cell lines, ESR1 and NAT1 exhibited increased expression in the ER + samples compared with in the ERsamples. NAT2 RNA expression did not significantly vary in breast cancer cell lines when comparing ER + and ERsamples, but was significantly increased in ER + primary breast tumor samples compared with in ERprimary breast tumor samples, although the difference was small. This finding suggested that the dysregulation of NAT1 and ESR1 during tumorigenesis may share similar mechanisms; however, NAT2 does not.
ESR1, NAT1 and NAT2 RNA expression were each increased in primary breast tumor samples compared with normal breast tissue samples although the significance and fold-change of NAT2 were smaller than that of ESR1 and NAT1. Additionally, for all genes, more widely spread expression was observed in the primary breast tumor samples compared with normal breast tissue. These data suggested that expression of all three genes may become modified during breast cancer tumorigenesis; however, the expression of NAT1 and ESR1 appear to be dysregulated to a greater extent. As recently reviewed (54), the role of NAT2 in breast cancer etiology is considered to be due to its effects on carcinogen metabolism. The present study suggested that the role of NAT2 in breast cancer is less likely a product of cell transformation, as the expression levels of NAT2 between normal and tumor tissues exhibited smaller variance than the expression levels of NAT1 and ESR1. NAT1 N-acetylation activity has been reported in normal breast tissue and breast tumor tissue (34,(36)(37)(38)(39)(40), whereas NAT2 N-acetylation activity has not been observed as consistently; when NAT2 activity is observed the activity is much lower than that of NAT1 activity (34,38,39). Wakefield et al profiled NAT1 expression and activity in seven breast cancer cell lines (MCF-7, T47D, ZR-75-1, Cal51, MDA-MB-231, MDA-MB-437 and MDA-MB-453); NAT1 mRNA and activity was observed in all seven cell lines (28); however, NAT2 expression and activity were not co-investigated. The high degree of association between the previously reported NAT1 N-acetylation activity and the NAT1 RNA expression of the same seven breast cancer cell lines suggested that NAT1 RNA expression is highly reflective of NAT1 N-acetylation activity. Gene expression is not always predictive of enzyme activity, due to the numerous regulatory mechanisms that can occur between RNA expression and protein function; however, these results suggested that RNA expression of NAT1 may serve as an appropriate predictor of NAT1 N-acetylation activity. Further studies with an increased number of breast cancer cell lines in which NAT1 N-acetylation activity has been measured are required to confirm this hypothesis. Additionally, further studies are required to determine association between NAT2 RNA expression and NAT2 N-acetylation activity.
The CCLE and TCGA repositories offer a wealth of publicly available data. The present study utilized this data to analyze and annotate the previously undefined relationships between NAT1, NAT2 and ESR1 in breast cancer cell lines, primary breast tumors and normal breast tissue. The results demonstrated that NAT1 and NAT2 RNA were expressed in normal breast tissue and primary breast tumor tissue; however, NAT1 RNA expression was much higher than NAT2. The expression of NAT1 and NAT2 were found to be associated; however, the magnitude was lower than that observed between NAT1 and ESR1 in the primary breast tumors and normal breast tissue. Additionally, although the association between NAT1 and NAT2 was slightly exaggerated in the breast cancer cell lines dataset, the cell lines generally reflected the NAT1 and NAT2 expression profiles of the primary breast tumors investigated. The present study demonstrated that while NAT1 and ESR1 expression were moderately associated in all datasets included in this study, NAT2 and ESR1 expression were associated at a lower magnitude, particularly in the primary breast tumor samples.
NAT1 and ESR1 expression were increased in primary breast tumor samples compared with normal breast tissue samples, and were increased in ER + primary breast tumors compared with ERprimary breast tumors. NAT2 expression was slightly increased in primary breast tumor samples compared with normal breast tissue samples and in ER + primary breast tumors compared with ERprimary breast tumors. Although NAT1 and NAT2 are both implicated in breast cancer, the majority of previous breast cancer studies have investigated each isozyme individually. The present study suggested that both isozymes should be considered in each study, since both are expressed in breast tissues. Defining the association between NAT1, NAT2 and ESR1 is of great importance, as modification of NAT1 is currently being studied for breast cancer prevention (20,21,55,56).
Novartis Institutes for Biomedical Research and its Genomics Institute of the Novartis Research Foundation. Information about TCGA and CCLE can be found at http://cancergenome.nih.gov and https://portals.broadinstitute.org/ccle, respectively. Those who carried out the original analysis and collection of the TCGA and CCLE data bear no responsibility for the further analysis or interpretation of it presented in this manuscript. Results of this study represent partial fulfillment of Samantha M. Carlisle's PhD in Pharmacology and Toxicology from the University of Louisville. The authors thank Mr. Patrick J. Trainor, MS, MA (Department of Medicine, University of Louisville, Louisville, KY, USA) for his careful review of the manuscript and insights.

Funding
The present study was partially supported by US Public Health Service grants (grant nos. T32-ES011564 and R25-CA134283).

Availability of data and materials
The CCLE data have been deposited in the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) using accession number GSE36139 and are also available at http:// www.broadinstitute.org/ccle. TCGA data portal can be accessed at https://portal.gdc.cancer.gov/ or via FirebrowseR, an R client to the Broad Institute's RESTful Firehose Pipeline.

Authors' contributions
SMC designed the study, retrieved and analyzed all data, and prepared all figures in partial fulfilment of her PhD dissertation carried out under the direction of DWH. Both authors drafted the manuscript. DWH reviewed, modified and approved the final manuscript.

Ethics approval and consent to participate
Not applicable.

Patient consent for publication
Not applicable.