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S13058-019-1150-Z.Pdf Wang et al. Breast Cancer Research (2019) 21:64 https://doi.org/10.1186/s13058-019-1150-z RESEARCHARTICLE Open Access Nicotinamide N-methyltransferase enhances chemoresistance in breast cancer through SIRT1 protein stabilization Yanzhong Wang1,2,3,4†, Jin Zeng1,3†, Weiping Wu1,5, Shuduo Xie3,6, Haitao Yu1,3, Guoli Li1,3, Tao Zhu3,7, Fengying Li1,3, Jie Lu1,3, Gavin Y. Wang4,8, Xinyou Xie1,2,3* and Jun Zhang1,2,3* Abstract Background: Nicotinamide N-methyltransferase (NNMT) is overexpressed in various human tumors and involved in the development and progression of several carcinomas. In breast cancer, NNMT was found to be overexpressed in several cell lines. However, the clinical relevance of NNMT in breast cancer is not yet clear. Methods: NNMT expression in breast carcinoma was examined by immunohistochemistry, and then, its relationship with patient clinicopathological characteristics was analyzed. The effects of NNMT on chemoresistance in breast cancer cells were assessed by cell viability, colony formation, and apoptosis assay. The NNMT, SIRT1, p53, and acetyl-p53 proteins, which are involved in NNMT-related chemoresistance, were examined by Western blotting. The SIRT1 mRNA was examined by real-time PCR, and its activity was measured by using the SIRT1 deacetylase fluorometric reagent kit. Results: NNMT expression was significantly higher (53.9%) in breast carcinoma than in paracancerous tissues (10.0%) and breast hyperplasia (13.3%). A high level of NNMT expression correlated with poor survival and chemotherapy response in breast cancer patients who received chemotherapy. Ectopic overexpression of NNMT significantly inhibited the apoptotic cell death and suppression of colony formation induced by adriamycin and paclitaxel. Mechanistic studies revealed that NNMT overexpression increased SIRT1 expression and promoted its activity. Either inhibition of SIRT1 by EX527 or knockdown of SIRT1 by siRNA could reverse NNMT-mediated resistance to adriamycin and paclitaxel, which suggests that SIRT1 plays a critical role in NNMT-related chemoresistance in breast cancer. Conclusions: The results of this study demonstrate a novel correlation between the NNMT expression level and patient survival, suggesting that NNMT has the potential to become a new prognostic biomarker to predict the treatment outcomes of the clinical chemotherapy in breast cancer. Moreover, targeting NNMT or downstream SIRT1 may represent a new therapeutic approach to improve the efficacy of breast cancer chemotherapy. Keywords: Breast cancer, NNMT, Chemoresistance, Survival, Chemotherapy response, SIRT1 Background overcome resistance and improve the efficacy of Breast cancer (BC) is the leading cause of cancer death chemotherapy. in women worldwide. Chemotherapy is an important ad- Nicotinamide N-methyltransferase (NNMT), a phase juvant for breast cancer treatment, but resistance is a II metabolizing enzyme, mainly catalyzes the methyla- major obstacle for chemotherapy in some patients. Com- tion of nicotinamide into 1-methylnicotinamide (MNA) bination treatment targeting molecules that contribute and other pyridines into pyridinium ions [1], and it is in- to chemoresistance is an important approach to volved in the biotransformation of many drugs and xe- nobiotics [2]. In 1984, Seifert R was the first to confirm * Correspondence: [email protected]; [email protected] † that alterations in NNMT activity are involved in the de- Yanzhong Wang and Jin Zeng contributed equally to this work. velopment and progression of carcinoma in vivo [3]. A 1Department of Clinical Laboratory, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, 3 East Qingchun Road, Hangzhou 310016, large number of subsequent studies demonstrated that Zhejiang, People’s Republic of China NNMT is aberrantly expressed and associated with a Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. Breast Cancer Research (2019) 21:64 Page 2 of 17 poor prognosis in various cancers, such as colorectal Cell line cancer [4], gastric cancer [5, 6], hepatocellular carcin- Human SK-BR-3 and MCF7 cell lines, which have low oma [7], and lung cancer [8]. Other studies have shown NNMT expression, and the MDA-MB-231 cell lines, that NNMT affects the proliferative, migratory, invasive, which have high NNMT expression, were obtained from and differentiation profiles of various cancers [9–11]. Cell Bank at the Chinese Academy of Sciences (Shang- Furthermore, NNMT overexpression has recently been hai, China) and cultured in DMEM (Gibco, Grand Is- found to be associated with chemotherapy resistance. land, NY, USA). The authenticity of the three cell lines After analyzing the correlation between the was verified using STR. All media were supplemented cancer-related genes and 99 anti-tumor drugs with with 10% fetal bovine serum (Gibco, Long Island, NY, known molecular mechanisms, Hsu et al. found that the USA), 100 U/ml penicillin (Sigma-Aldrich, St. Louis, NNMT expression level might be related to the sensitiv- MO, USA), and 100 mg/ml streptomycin (Sigma-Al- ity to chemotherapeutic drugs [12]. Yu et al. reported drich, St. Louis, MO, USA), and the cells were main- that NNMT knockdown PANC-1 cells were much less tained at 37 °C in a humidified 5% CO2 incubator. resistant to rapamycin as well as glycolytic inhibitor 2-deoxyglucose, whereas NNMT-overexpressing cells Human tissue specimens and patient clinical information showed the opposite effects [11]. We also have previ- This study was approved by the Human Research Ethics ously reported that NNMT overexpression inhibits the Committee of Sir Run Run Shaw Hospital (Hangzhou, activation of ASK1-p38 pathway via MNA production, China). As the initial treatment, total 165 which results in a decrease in the apoptosis induced by treatment-naive patients with breast cancer received 5-fluorouracil (5-FU) to enhance resistance in colorectal mastectomy from Oct 1, 2000, to Dec 31, 2006, 82 of cancer cells [13]. These reports suggested that NNMT whom had a chemotherapy efficacy record at Sir Run might be involved in the resistance to chemotherapy and Run Shaw Hospital (Hangzhou, China) and were there- thus serve as a potential target for combination therapy. fore included in this study. The diagnoses of breast can- Therefore, we investigated the role of NNMT in breast cer were confirmed by postoperative pathological cancer chemotherapy, which might be beneficial for im- results. proving chemotherapeutic efficacy in breast cancer. The clinical characteristics of 165 cancer patients were At the beginning of this study, we found that NNMT extracted from their medical record, including age, gen- was upregulated in breast carcinomas of patients who der, tumor diameter, TNM stage, ER, HER-2, PR, Ki-67, were undergoing mastectomy by immunohistochemistry and chemotherapy response judged by the revised on tissue microarray (p < 0.001). After correlation with RECIST guideline (version 1.1). Eighty-two patients with the clinicopathological characteristics of 82 patients with breast cancer received the chemotherapy mainly using their chemotherapy efficacy record, NNMT overexpres- CMF (cyclophosphamide + methotrexate + fluorouracil) sion was found to be associated with a shorter survival and FEC-P (fluorouracil + epirubicin + cyclophospha- and reduced chemotherapy efficacy (p < 0.05). We then mide + paclitaxel) regimens, which accounted for more confirmed that NNMT overexpression significantly en- than 90% of the patients. These patients were followed hanced resistance to adriamycin (ADM) and paclitaxel up, and the OS were calculated from the date of surgical (PTX) in BCs. Furthermore, we demonstrated that treatment to the date of death or last follow-up. NNMT overexpression attenuated the apoptosis that was induced by ADM and PTX to enhance the resist- ance through SIRT1 protein stabilization in BCs. Immunohistochemistry analysis (IHC) on paraffin- embedded tissue array The tissue microarray block was cut into 4-μm sections, Methods and immunohistochemical staining was performed. Drugs and antibodies Briefly, the sections were first deparaffinized and hy- Adriamycin (ADM) and 1-methylnicotinamide (MNA) drated. After antigen retrieval with 0.01 M citrate buffer were obtained from Sigma-Aldrich (Sigma-Aldrich, St. (pH 6.0) and microwave heat induction, the sections Louis, MO, USA), and paclitaxel (PTX) and the select- were treated with 3% hydrogen peroxide for 10 min. ively SIRT1 inhibitor EX527 were obtained from Selleck NNMT were detected using mouse monoclonal Chemicals (SelleckChemicals, Houston, TX, USA). The anti-human NNMT antibody (dilution 1:10000). After anti-SIRT1, anti-β-actin, and anti-acetyl-p53 were all ob- secondary antibody staining, diaminobenzidine was used tained from Cell Signaling Technology (CST, Beverly, as the chromogen for 3 min, and then, the nuclei were MA, USA). The mouse anti-NNMT monoclonal anti- counterstained with
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