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Upregulation of cytidine deaminase in NAT1 knockout breast cancer cells - PubMed

Upregulation of cytidine deaminase in NAT1 knockout breast cancer cells

Kyung U Hong et al. J Cancer Res Clin Oncol. 2023 Jul.

Abstract

Purpose: Arylamine N-acetyltransferase 1 (NAT1), a phase II metabolic enzyme, is frequently upregulated in breast cancer. Inhibition or depletion of NAT1 leads to growth retardation in breast cancer cells in vitro and in vivo. A previous metabolomics study of MDA-MB-231 breast cancer cells suggests that NAT1 deletion leads to a defect in de novo pyrimidine biosynthesis. In the present study, we observed that NAT1 deletion results in upregulation of cytidine deaminase (CDA), which is involved in the pyrimidine salvage pathway, in multiple breast cancer cell lines (MDA-MB-231, MCF-7 and ZR-75-1). We hypothesized that NAT1 KO MDA-MB-231 cells show differential sensitivity to drugs that either inhibit cellular pyrimidine homeostasis or are metabolized by CDA.

Methods: The cells were treated with (1) inhibitors of dihydroorotate dehydrogenase or CDA (e.g., teriflunomide and tetrahydrouridine); (2) pyrimidine/nucleoside analogs (e.g., gemcitabine and 5-azacytidine); and (3) naturally occurring, modified cytidines (e.g., 5-formyl-2'-deoxycytidine; 5fdC).

Results: Although NAT1 KO cells failed to show differential sensitivity to nucleoside analogs that are metabolized by CDA, they were markedly more sensitive to 5fdC which induces DNA damage in the presence of high CDA activity. Co-treatment with 5fdC and a CDA inhibitor, tetrahydrouridine, abrogated the increase in 5fdC cytotoxicity in NAT1 KO cells, suggesting that the increased sensitivity of NAT1 KO cells to 5fdC is dependent on their increased CDA activity.

Conclusions: The present findings suggest a novel therapeutic strategy to treat breast cancer with elevated NAT1 expression. For instance, NAT1 inhibition may be combined with cytotoxic nucleosides (e.g., 5fdC) for breast cancer treatment.

Keywords: Arylamine N-acetyltransferase; Breast cancer; Chemotherapeutic; Cytidine analogs; Cytidine deaminase; Pyrimidine biosynthesis.

© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.

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Conflict of interest statement

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Figures

Fig. 1
Fig. 1. Deletion of NAT1 leads to increases in CDA mRNA, protein, and activity levels in breast cancer cell lines

NAT1 KO cell lines, previously generated using three breast cancer cell lines (MDA-MB-231, MCF-7, and ZR-75–1), and their corresponding parental cells were analyzed for CDA (cytidine deaminase) expression. A and B, CDA mRNA expression. Total RNA isolated from the indicated cell lines were analyzed for the relative level of CDA mRNA using RT-qPCR. N = 3 per group. C, Western blot for CDA protein. Total cell lysates from the indicated cell lines were separated on an SDS-PAGE gel and blotted with a CDA-specific antibody. α-tubulin served as an internal, loading control. D, CDA enzyme activity assay. Lysates from the indicated cell lines were incubated with cytidine (substrate), and the formation of uridine (product) was quantified using HPLC. N = 3 per group. P, parental; 2, NAT1 KO2; 5, NAT1 KO5. The graphs show mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The p values are for the difference between the parental and each of the NAT1 KO cell lines.

Fig. 2.
Fig. 2.. Correlation between NAT1 and CDA transcript levels in human tumors and normal tissues

The dot plots show the mRNA level (expressed in log2 of transcripts per kilobase million [TPM]) of NAT1 (x-axis) and the corresponding mRNA level of CDA (y-axis) in the same individual samples. A, NAT1 vs. CDA expression in TCGA breast tumors only. B, NAT1 vs. CDA expression in all tumors in TCGA database. C, NAT1 vs. CDA expression in normal human tissues from GTEx database. R, Pearson correlation coefficient. The analysis was done using tools available at GEPIA (Tang et al. 2017).

Fig. 3
Fig. 3

Chemical structures of the drugs tested in the present study

Fig. 4
Fig. 4. Sensitivity of parental vs. NAT1 KO MDA-MB-231 cells to inhibitors of de novo pyrimidine synthesis and CDA

To compare the sensitivity of the parental and two NAT1 KO MDA-MB-231 cells (KO2 and KO5) to inhibitors of pyrimidine biosynthesis or salvage pathway, the cells were treated with the indicated concentrations of teriflunomide and leflunomide (inhibitors of dihydro-orotate dehydrogenase, a key enzyme in the de novo pyrimidine synthesis pathway) or tetrahydrouridine and zebularine (inhibitors of CDA) for 3 days. Following the treatment, cell viability was measured using alamarBlue assay and expressed as ‘relative cell viability’ (relative to the untreated control group). Drug concentrations are shown in a log scale. N = 4 per concentration. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The p values are for the difference between the parental and each of the NAT1 KO cell lines. Red asterisks indicate the significance of the difference between parental and KO2, while blue asterisks indicate the significance of the difference between parental and KO5.

Fig. 5
Fig. 5. Sensitivity of the parental vs. NAT1 KO MDA-MB-231 cells to pyrimidine/nucleoside analogs

To compare the sensitivity of the parental (P) and NAT1 KO MDA-MB-231 cells (KO2 and KO5) to chemotherapeutic drugs that are subject to metabolism by CDA, the cells were treated with the indicated concentrations of gemcitabine, 5-azacytidine, cytosine-β-D-arabinofuranoside (Ara-C) or capecitabine (see Fig. 3 for their structures) for 7 days. Following the treatment, cell viability was measured using alamarBlue assay and expressed as ‘relative cell viability’ (relative to the untreated control group). Drug concentrations are shown in a log scale. N = 4 per concentration. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The p values are for the difference between the parental and each of the NAT1 KO cell lines. Red asterisks indicate the significance of the difference between parental and KO2, while blue asterisks indicate the significance of the difference between parental and KO5.

Fig. 6
Fig. 6. Sensitivity of the parental vs. NAT1 KO breast cancer cells to naturally occurring, epigenetically modified cytidines

To compare the sensitivity of the parental and NAT1 KO breast cancer cell lines to naturally occurring, modified cytidines, the cells were treated with the indicated concentrations of 5fdC (5-formyl-2’-deoxycytidine) (panels A, B, D, and E) or 5hmdC (5-hydroxymethyl-2’-deoxycytidine) (panel C) for 7 days. Following the treatment, cell viability was measured using alamarBlue assay and expressed as ‘relative cell viability’ (relative to the untreated control group). A-C, Parental vs. NAT1 KO (KO2 and KO5) MDA-MB-231 cells. D, Parental vs. NAT1 KO (KO2 and KO5) MCF-7 cells. E. Parental vs. NAT1 KO (KO2) ZR-75–1 cells. Drug concentrations are shown in a log scale for panels A and C only. N = 4 per concentration. *, p < 0.05; **, p < 0.01; ***, p < 0.001. The p values are for the difference between the parental and each of the NAT1 KO cell lines. Red asterisks indicate the significance of the difference between parental and KO2, while blue asterisks indicate it between parental and KO5.

Fig. 7
Fig. 7. Increased sensitivity of NAT1 KO MDA-MB-231 cells to 5fdC is dependent on CDA activity.

Parental and two NAT1 KO MDA-MB-231 cell lines (KO2 and KO5) were treated with the indicated concentrations of 5fdC (5-formyl-2’-deoxycytidine) alone (white bars) or co-treated with 5fdC and a CDA inhibitor, tetrahydrouridine (THU; 60 μM) (gray bars) for 3 days. Following the treatment, cell viability was measured using alamarBlue assay and expressed as ‘relative cell viability’ (relative to the control group, i.e., 0 μM 5fdC without THU). N = 4 per group. ***, p < 0.001. The p values are for the difference between the 5fdC only and 5fdC + THU treatment groups within each cell line.

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References

    1. Bar-Or A, Pachner A, Menguy-Vacheron F, et al. (2014) Teriflunomide and Its Mechanism of Action in Multiple Sclerosis. Drugs 74:659–674. 10.1007/s40265-014-0212-x - DOI - PMC - PubMed
    1. Billam M, Sobolewski MD, Davidson NE (2010) Effects of a novel DNA methyltransferase inhibitor zebularine on human breast cancer cells. Breast Cancer Res Treat 120:581–592. 10.1007/s10549-009-0420-3 - DOI - PMC - PubMed
    1. Brown KK, Spinelli JB, Asara J, Toker A (2017) Adaptive Reprogramming of De Novo Pyrimidine Synthesis is a Metabolic Vulnerability in Triple-Negative Breast Cancer. Cancer Discov 7:391–399. 10.1158/2159-8290.CD-16-0611 - DOI - PMC - PubMed
    1. Carlisle SM, Hein DW (2018) Retrospective analysis of estrogen receptor 1 and N-acetyltransferase gene expression in normal breast tissue, primary breast tumors, and established breast cancer cell lines. Int J Oncol 53:694–702. 10.3892/ijo.2018.4436 - DOI - PMC - PubMed
    1. Carlisle SM, Trainor PJ, Doll MA, Hein DW (2021) Human Arylamine N-Acetyltransferase 1 (NAT1) Knockout in MDA-MB-231 Breast Cancer Cell Lines Leads to Transcription of NAT2. Front Pharmacol 12:803254. 10.3389/fphar.2021.803254 - DOI - PMC - PubMed

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