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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 - PubMed

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

Samantha M Carlisle et al. Int J Oncol. 2018 Aug.

Abstract

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.

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Figures

Figure 1
Figure 1

Scatterplot and correlation matrices for NAT1, NAT2, and ESR1. Associations between NAT1, NAT2, and ESR1 RNA expression were analyzed in breast cancer cell lines, primary breast tumor tissue, and normal breast tissue using the Spearman method. In the scatterplot matrices, each open circle represents a single sample and is color-coded according to ER status; pink circles, ER− samples; blue circles, ER+ samples; black circles, samples with unknown ER status. In the association matrices, boxes are labeled with the Spearman correlation coefficient (ρ) for each comparison and color reflects strength of association; dark blue represents high association, light blue represents low association, and white represents no association. (A) Scatterplot matrix of the association between NAT1, NAT2 and ESR1 RNA expression in breast cancer cell lines (n=57). (B) Correlation matrix between NAT1, NAT2 and ESR1 RNA expression in breast cancer cell lines (n=57). (C) Scatterplot matrix of the association between NAT1, NAT2 and ESR1 RNA expression in primary breast tumor samples (n=1,043 for NAT1 vs. ESR1, n=984 for NAT1 vs. NAT2 and NAT2 vs. ESR1). (D) Correlation matrix between NAT1, NAT2, and ESR1 RNA expression in primary breast tumor samples (n=1,043 for NAT1 vs. ESR1, n=984 for NAT1 vs. NAT2 and NAT2 vs. ESR1). (E) Scatterplot matrix of the association between NAT1, NAT2 and ESR1 RNA expression in normal breast tissue samples (n=99 for NAT1 vs. ESR1, n=92 for NAT1 vs. NAT2 and NAT2 vs. ESR1). (F) Correlation matrix between NAT1, NAT2 and ESR1 RNA expression in normal breast tissue samples (n=99 for NAT1 vs. ESR1, n=92 for NAT1 vs. NAT2 and NAT2 vs. ESR1). ER, estrogen receptor; ESR1, estrogen receptor 1; NAT1, arylamine N-acetyltransferase 1; NAT2, arylamine N-acetyltransferase 2.

Figure 2
Figure 2

NAT1 and NAT2 RNA expression in breast cancer cell lines, primary breast tumor samples, and normal breast tissue samples. Differences in gene expression between NAT1 and NAT2 in breast cancer cell lines, primary breast tumor tissue and normal breast tissue were statistically evaluated by Wilcoxon rank-sum test; ***P<0.001. Each dot represents a single sample and is color-coded according to ER status; pink dots, ER− samples; blue dots, ER+ samples; black dots, samples with unknown ER status. 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) NAT1 RNA expression was significantly higher than NAT2 RNA expression in the breast cancer cell lines. (B) NAT1 RNA expression was significantly higher than NAT2 RNA expression in the primary breast tumor samples. (C) NAT1 RNA expression was significantly higher than NAT2 RNA expression in the normal breast tissue samples. ER, estrogen receptor; IQR, interquartile range; NAT1, arylamine N-acetyltransferase 1; NAT2, arylamine N-acetyltransferase 2; RPKM, reads per kilobase of transcript per million mapped reads; RSEM, RNA-Seq by Expectation-Maximization.

Figure 3
Figure 3

ESR1, NAT1 and NAT2 RNA expression in breast cancer cell lines, primary breast tumor samples, and normal breast tissue stratified by ER status. Differences in the expression levels of ESR1, NAT1 and NAT2 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, ER− samples; 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 ER− breast cancer cell lines. NAT2 RNA expression was not significantly different in ER+ breast cancer cell lines compared with ER− breast 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. (B) ESR1, NAT1 and NAT2 RNA expression were significantly higher in ER+ samples compared with ER− samples in the primary breast tumor dataset. (C) ESR1, NAT1 and NAT2 RNA expression levels were not significantly different in ER+ samples compared with ER− samples in the normal breast tissue dataset. ER, estrogen receptor; IQR, interquartile range; ESR1, estrogen receptor 1; NAT1, arylamine N-acetyltransferase 1; NAT2, arylamine N-acetyltransferase 2; RPKM, reads per kilobase of transcript per million mapped reads; RSEM, RNA-Seq by Expectation-Maximization.

Figure 4
Figure 4

Comparison of ESR1, NAT1 and NAT2 RNA expression in normal breast tissue and primary breast tumor samples. Differences in gene expression of ESR1, NAT1 and NAT2 in normal breast tissue and primary breast tumor tissue were evaluated by Wilcoxon rank-sum test; ***P<0.001; *P<0.05. 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.

Figure 5
Figure 5

Association between NAT1 RNA expression and previously reported NAT1 N-acetylation activity in seven established breast cancer cell lines. NAT1 RNA expression from Cancer Cell Line Encyclopedia and previously reported NAT1 N-acetylation activity (28) in seven breast cancer cell lines were significantly associated (P<0.05; ρ=0.89). Dots represent a single cell line and are color-coded according to ER status: Pink dots, ER− samples; blue dots, ER+ samples. ER, estrogen receptor; NAT1, arylamine N-acetyltransferase 1; NAT2, arylamine N-acetyltransferase 2; PABA, p-aminobenzoic acid; RPKM, reads per kilobase of transcript per million mapped reads.

Figure 6
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, ER− samples; blue dots, ER+ samples; black dots, samples with unknown or conflicting ER status in the literature (–48). NAT1 and NAT2 RNA expression was significantly associated in breast cancer cell lines (P<0.0001, ρ= 0.64).

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References

    1. Hein DW. Molecular genetics and function of NAT1 and NAT2: Role in aromatic amine metabolism and carcinogenesis. Mutat Res. 2002:506–507. 65–77. - PubMed
    1. Hein DW, Doll MA, Fretland AJ, Leff MA, Webb SJ, Xiao GH, Devanaboyina US, Nangju NA, Feng Y. Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms. Cancer Epidemiol Biomarkers Prev. 2000;9:29–42. - PubMed
    1. Bendaly J, Zhao S, Neale JR, Metry KJ, Doll MA, States JC, Pierce WM, Jr, Hein DW. 2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline-induced DNA adduct formation and mutagenesis in DNA repair-deficient Chinese hamster ovary cells expressing human cytochrome 4501A1 and rapid or slow acetylator N-acetyltransferase 2. Cancer Epidemiol Biomarkers Prev. 2007;16:1503–1509. doi: 10.1158/1055-9965.EPI-07-0305. - DOI - PMC - PubMed
    1. Millner LM, Doll MA, Cai J, States JC, Hein DW. NATb/NAT1*4 promotes greater arylamine N-acetyltransferase 1 mediated DNA adducts and mutations than NATa/NAT1*4 following exposure to 4-aminobiphenyl. Mol Carcinog. 2012;51:636–646. doi: 10.1002/mc.20836. - DOI - PMC - PubMed
    1. Hein DW. Acetylator genotype and arylamine-induced carcinogenesis. Biochim Biophys Acta. 1988;948:37–66. - PubMed

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