Nicotinamide N-methyltransferase enhances paclitaxel resistance in ovarian clear cell carcinoma

Nicotinamide N-methyltransferase enhances paclitaxel resistance in ovarian clear cell carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Nicotinamide N-methyltransferase enhances paclitaxel resistance in ovarian clear cell carcinoma Ryoko Kikuchi-Koike, Masaru Sakamoto, Yuko Sasajima, Yuko Miyagawa, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4249856/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background. Growing evidence suggests that nicotinamide N-methyltransferase (NNMT), an S-adenosyl-L-methionine (SAM)-dependent cytosolic enzyme, plays an essential role in cancer progression. Recently, it was reported that NNMT is involved in methylation metabolism and tumorigenesis and is associated with poor prognosis in a number of cancers. It has also been reported that NNMT is overexpressed in the stroma of advanced high-grade serous carcinomas and may contribute to poor survival. The aim of this study was to identify novel biomarkers that predict resistance in paclitaxel-resistant advanced or recurrent ovarian clear cell carcinoma (OCCA) and to evaluate their clinicopathologic significance. Methods. Four OCCA cell lines (ES-2, KK, OVMANA, and OVTOKO) were divided into paclitaxel high and low sensitivity groups by WST-8 assay and fluorescence-labeled two-dimensional electrophoresis (2D-DIGE) was performed. Protein spots with different expression intensities in each drug-sensitive group were analyzed by mass spectrometry to identify the proteins. Results. NNMT was detected as a protein molecule upregulated in the paclitaxel-resistant group, and knockdown by NNMT siRNA increased paclitaxel sensitivity in the NNMT-expressing ovarian clear cell carcinoma cell lines OVTOKO and RMG1. Furthermore, in analysis of clinical tissue samples, no deaths were observed in 7 patients with low NNMT expression in the cytoplasm of cancer cells. Conclusions. High NNMT expression in the cytoplasm of cancer cells is associated with low sensitivity to paclitaxel in OCCA and may have prognostic implications; knockdown of NNMT expression also reduced paclitaxel efficacy. Therefore, targeted therapies that reduce cytoplasmic NNMT expression levels may increase the sensitivity of OCCA to paclitaxel. Ovarian clear cell adenocarcinoma chemoresistance paclitaxel nicotinamide N-methyltransferase 2D-DIGE Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Ovarian cancer is the fifth most common cause of cancer-related deaths in women after lung, breast, colorectal, and pancreatic cancers [ 1 ]. Ovarian clear cell adenocarcinoma (OCCA) is a rare and distinct histological type of epithelial ovarian cancer (EOC). Women diagnosed with OCCA are usually younger and at an earlier stage of cancer than those with the common high-grade serous adenocarcinoma histology [ 2 ]. The overall prognosis of OCCA is good because most cases are identified during stage I of cancer [ 3 ]. However, advanced and recurrent disease is associated with a poor prognosis and resistance to standard treatment [ 4 ]. While serous adenocarcinoma, the most common type of ovarian cancer, responds well to anticancer drug therapy, ovarian clear cell carcinoma is known to have an abysmal prognosis due to the low efficacy of anticancer drug therapy. Although there are many patients with this type of ovarian clear cell carcinoma in Asia, the number of patients with this type of cancer is relatively small in Europe and the United States, and no progress has been made in developing therapeutic agents. Patients with OCCA show a lower response rate to paclitaxel and carboplatin treatment regimens than the non-OCCA ovarian cancer group [ 5 ]. Improving the response rate of patients with OCCA to paclitaxel and carboplatin regimens may improve the prognosis of patients with OCCA. Aoki et al. reported that taxane-based chemotherapy was effective in patients with OCCA that were positive for beta-tubulin III [ 6 ]. Ho et al. reported that restoration of HIN1 expression reversed paclitaxel resistance in OCCA [ 7 ]. Yet, the molecules involved in the efficacy of taxane-based chemotherapy for OCCA have not been identified. A retrospective analysis of a multicenter, Phase 3, randomized, controlled trial (JGOG3017/GCIG) comparing TC (carboplatin and paclitaxel) and CPT-P (irinotecan and cisplatin) therapy in patients with recurrent or persistent OCCA found that patients with platinum-resistant relapse had a significantly shorter median post-progression survival (PPS) compared to those with platinum-sensitive relapse [ 8 ]. Combination therapy with gemcitabine, cisplatin, and bevacizumab (GP + Bev) has also been reported to be promising for advanced OCCA [ 9 ]. The identification of molecules involved in paclitaxel resistance in OCCA may provide an opportunity to improve the efficacy of paclitaxel and carboplatin regimens in OCCA. Therefore, we aimed to analyze the effect of paclitaxel treatment on the OCCA cell lines ES-2, KK, OVMANA, and OVTOKO, and the associated molecular changes using fluorescence-labeled two-dimensional differential gel electrophoresis (2D-DIGE). Material and Methods Cell lines and primary tumor samples OVTOKO and OVMANA cell lines were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan), ES-2 from the American Type Culture Collection, and KK from the National Defense Medical College [ 10 ]. Primary tumor samples were obtained during surgery from 39 patients treated at Teikyo University Hospital in Tokyo, with written consent obtained from each patient after approval was approved by the Clinical Ethics Committee of the Medical Faculty at Teikyo University (approval number: 13-003-4 on 22 October 2020). The study adheres to the provisions specified in the Declaration of Helsinki. Proliferation assay The effects of paclitaxel on cell proliferation were evaluated using a WST-8 assay (Dojindo, Kumamoto, Japan). The assay can be used to evaluate cell viability in cell proliferation assays. WST-8 is reduced by dehydrogenase activity in cells to produce a water-soluble yellow-colored formazan dye formed by NADH produced in the mitochondria. The amount of formazan dye produced is directly proportional to the number of living cells. The protocol is described briefly as follows. Cells were seeded in 24-well plates and pre-incubated at 37°C for 24 h to enable cell attachment. At 72 h after drug treatment, 50 µL of WST-8 solution was added to each well and incubated at 37°C for 0.5–4 h (the reaction time was adjusted according to the cell line). The augmentation of enzyme activity increased the amount of formazan dye, which was quantified using a microplate reader (Bio-Rad, Tokyo, Japan) by measuring absorbance at 450–550 nm. This procedure was repeated at least three times. Tissue immunohistochemistry and immunofluorescence Formalin-fixed paraffin-embedded (FFPE) tissue specimens were sliced at 4 µm thickness, deparaffinized in xylene, and rehydrated using graded ethanol solutions. After activation in citric acid buffer at 98°C for 40 min, the slides were stained with anti-NNMT (Santa Cruz, Tokyo, Japan G-4; 1:100) antibodies and processed using the EnVision FLEX Visualization System (Agilent Technologies). Samples were counterstained with Mayer’s hematoxylin (131–09665, Fujifilm, Tokyo, Japan). Tissue microarray analysis We analyzed NNMT expression using a tissue microarray (TMA) for 39 patients with ovarian clear cell adenocarcinoma who underwent surgery at Teikyo University Hospital between January 2003 and December 2012. Detailed information about the clinical characteristics was obtained after a retrospective review of the medical records. NNMT immunohistochemical reactivity was scored without knowledge of the clinical outcomes by two observers (R.K. and Y.S.). Each sample was scored based on the percentage of positive cells in each compartment (0, no staining; 1, < 30%; 2, 30–50%; 3, ≥ 50%). The staining intensity was similar across all samples. Expression was considered ‘low’ if the cytoplasmic staining intensity was 0 or 1 and ‘high’ if scores of 2 or 3 were obtained. The analysis was limited to patients with ovarian clear cell carcinoma (N = 39). Kaplan–Meier survival curves and log-rank test were used to compare the difference and overall survival were generated using BellCurve for Excel. Fluorescence-labeled two-dimensional differential gel electrophoresis (2D-DIGE) Proteins were labeled using the CyDye DIGE Fluor minimal dye (GE Healthcare), as per the manufacturer’s instructions. Briefly, 50 mg of a sample mixture extracted from the ovarian clear cell cancer cell lines (OVTOKO, OVMANA, ES-2, and KK) was adjusted to pH 8.5 using 50 mM NaOH, and samples were labeled with 400 pmol Cy5 as a control or with 400 pmol Cy3. Fluorescence labeling was performed on ice in the dark for 30 min and the reaction was subsequently quenched with 1 mL of 10 mM lysine (Sigma-Aldrich, St. Louis, USA) for 10 min. Each preparation was treated with two sample buffers containing 7 M urea, 2 M thiourea, 4% CHAPS, 1% immobilized pH gradient (IPG)-buffer with a pH range of 4–7, and 2% dithiothreitol (DTT) according to the manufacture’s recommendations. The final volume was adjusted to 260 mL using rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.5% IPG buffer with a pH range of 4–7, and 0.2% DTT). The mixture of proteins labeled with Cy3 and Cy5 was applied to Immobiline DryStrips (pH 4–7, 13 cm) (Cytiva, Tokyo, Japan) and focused on a PROTEAN i12 IEF system (Bio-Rad, Tokyo, Japan). Focused IPG strips were equilibrated and loaded onto 12.5% SDS-polyacrylamide gels (30% acrylamide, 1.5 M Tris-HCl pH 8.8, 10% SDS, 10% ammonium persulfate, and 10% TetraMethylEthyleneDiamine (TEMED)) using low-fluorescence glass plates on an SE 600 Ruby system (GE Healthcare). All electrophoresis procedures were performed in the dark. After SDS-PAGE, gels were scanned using a Pharos FX System (Bio-Rad) with appropriate excitation/emission wavelengths specific for Cy3 (532/605 nm) and Cy5 (635/695 nm). Scanned images were analyzed using the PDQuest Advanced Version 8.0 software (Bio-Rad). PDQuest was used to identify spots with higher or lower protein expression in ES-2 and KK, and OVMANA and OVTOKO, respectively. Five spots were identified. Mass spectrometric analysis showed that NNMT was present in one of the spots. The spot containing NNMT had higher protein levels in OVMANA and OVTOKO than in KK and ES-2. For further confirmation, ES-2 and OVMANA, ES-2 and OVTOKO, KK and OVMANA, KK, and OVTOKO were labeled with Cy3, Cy5, Cy5, and Cy3, respectively. The protein expression level of the spot containing NNMT was higher in OVMANA than ES-2, OVTOKO than ES-2, OVMANA than KK, and OVTOKO than KK. Peptide mass fingerprinting (PMF) All chemicals used in this study were of analytical grade. The compounds 4-Sulfophenyl isothiocyanate, a-cyano-4-hydroxycinnamic acid (CHCA), sodium bicarbonate, and ammonium bicarbonate were purchased from Sigma (St. Louis, MO, USA). For protein identification by peptide mass fingerprinting, protein spots were excised, digested with trypsin (Promega, Madison, WI), mixed with α-cyano-4-hydroxycinnamic acid in 50% acetonitrile /0.1% TFA, and subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis (Microflex LRF 20, Bruker Daltonics, MA, USA), as described by Fernandez et al. (Electrophoresis 19:1036–1045). Spectra were collected from 300 shots per spectrum over an m/z range of 600–3000 and calibrated by two-point internal calibration using trypsin auto-digestion peaks ( m/z 842.5099 and 2211.1046). The peak list was generated using Flex Analysis 3.0. The threshold used for peak-picking was as follows: 500 for the minimum resolution of monoisotopic mass and 5 for S/N. The search program MASCOT, developed by Matrixscience ( http://www.matrixscience.com/ ), was used for protein identification using peptide mass fingerprinting. The following parameters were used for the database search: trypsin as the cleaving enzyme, maximum of one missed cleavage, iodoacetamide (Cys) as a complete modification, oxidation (Met) as a partial modification, monoisotopic masses, and a mass tolerance of ± 0.1 Da. The PMF acceptance criterion included the probability scores. Western blot analysis Equal amounts of protein were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membranes were blocked, the primary antibodies were added, and the membranes were incubated with secondary antibodies. Signals were detected using an Image Quant LAS 4000 Mini instrument (GE Healthcare, Wauwatosa, WI, USA). siRNA We transfected NNMT siRNA (sc-61213; Santa Cruz Biotechnology) into the OVMANA, RMG1, and ES-2 cell lines cultured in 6-well plates using Lipofectamine RNAi MAX Reagent (13778; Invitrogen, Tokyo, Japan) and Opti-MEM Reduced Serum Medium (31985070; Thermo Fisher Scientific, Tokyo, Japan). Control siRNA-A (sc-37007; Santa Cruz Biotechnology, Tokyo, Japan) was used as a negative control. The knockdown effect was tested by western blotting using an anti-NNMT antibody. Expression plasmid transfection We transfected the NNMT Expression Plasmid DNA (RC200641; OriGene Technologies, Inc., MD, USA) into the ES-2 cell line that was cultured in 6-well plates using Lipofectamine 2000 Transfection Reagent (11668019; Thermo Fisher Scientific) and Opti-MEM Reduced Serum Medium (31985070; Thermo Fisher Scientific Tokyo, Japan). The pCMV vector was used as a negative control for all experiments. The cells were incubated for 2 weeks in a medium containing G418 (1000 µg/mL; Thermo Fisher Scientific, Tokyo, Japan). We also established stable control clones expressing NNMT in these cell lines. Drug-resistant clones were further incubated in a medium with puromycin (58-58-2; Sigma-Aldrich, Tokyo, Japan) and tested for the knockdown effect by western blotting using the NNMT antibody. Statistical analysis The Student t -test was used to determine the statistical significance of the differences between the comparison groups in vitro. Error bars represent the mean ± standard error of the mean. The relationship between NNMT expression and clinicopathological characteristics was analyzed using Pearson’s χ2 test. Data is equally distributed, and survival rates were calculated using the Kaplan–Meier method and log-rank test. Results Proliferation of OCCA cell lines was differentially inhibited by paclitaxel Using the WST-8 assay, we showed that paclitaxel inhibited the proliferation of OVTOKO, OVMANA, ES-2, and KK cells in a dose-dependent manner. Paclitaxel inhibited the proliferation of OVTOKO and OVMANA cells more than that of ES-2 and KK (IC50 = 0.0826 ± 0.0054, 0.0703 ± 0.0092, 0.0127 ± 0.0027, 0.0047 ± 0.0002, respectively) (Fig. 1 ). 2D-DIGE analysis of OCCA cell lines and mass spectrometric analysis The mixture of OVTOKO, OVMANA, KK, and ES-2 cellular proteins was labeled with Cy5 and used as an internal control. The OVTOKO, OVMANA, KK, and ES-2 proteins from individual cell lines were labeled with Cy3. The mixture of the proteins labeled with Cy3 and Cy5 was analyzed using four 2D-DIGE gels. A total of 130 protein spots were detected from the mixture of proteins from OVTOKO, OVMANA, KK, and ES-2 cells on each gel using PDQuest Advanced software, and all spots on each gel were matched. A total of 128 protein spots were detected, and the spots for OVTOKO, OVMANA, KK, and ES-2 cells, and all spots on each gel were matched using the PDQuest Advanced software. We detected four spots that showed greater intensity in OVTOKO and OVMANA than in KK and ES-2 cells, and one spot that showed greater intensity in KK and ES-2 than in OVTOKO and OVMANA cells by creating analysis sets using PDQuest Advanced software. The spots and quantitative graphs for each spot are shown in Fig. 2 A. The red bars in the quantitative graph show the number of spots from ES-2, KK, OVMANA, and OVTOKO cells (from left to right) and the green bars show the number of spots from the mixture of ES-2, KK, OVMANA, and OVTOKO cells on each gel (Fig. 2 A). The three spots were detected via Coomassie Brilliant Blue staining. The samples were next subjected to mass spectrometric analysis. In one of the three spots, an unnamed protein with a molecular weight of ~ 36 KDa and a pI of 5.88 was detected in a MASCOT search with the top score of 161 (data not shown). In another one of the 3 spots with a molecular weight of ~ 33 KDa and pI 4.99, we detected Mixture 1 (glyceraldehyde-3-phosphate dehydrogenase, chain A, annexin A2, and chain A, crystal structure of human Mu_crystallin at 2.6 Å) at the top score 350 (data not shown). In another one of the 3 spots with a molecular weight of ~ 30 KDa and pI 5.56, we detected NNMT via a Mascot search with a top score of 159 (Fig. 3 A). Figure 3 B shows the list of digested peptides derived from the spot, which was identified as NNMT in a MASCOT search. Supplementary Fig. 1B shows the NNMT protein sequence coverage of the digested peptides. We confirmed that the spot containing NNMT had higher protein levels in OVMANA than in ES-2 (Fig. 2 B), in OVMANA than in KK, in OVTOKO than in ES-2, and in OVTOKO than in KK cells (Supplementary Fig. 2A, B, C). Validation of the 2D-DIGE results via western blot analysis We detected NNMT in OVTOKO and OVMMANA cells, but not in ES-2 and KK cells, using western blot analysis. Thus, the 2D-DIGE results were validated using western blot analysis (Fig. 3 B). Association between the expression level of NNMT and clinicopathologic characteristics in cases with primary tumors To further clarify the clinical significance of NNMT levels in OCCA, the expression level of the protein in primary OCCA tissues was evaluated by immunohistochemistry using an NNMT-specific antibody. Although a few OCCA specimens frequently showed high levels of NNMT in the cytoplasm (Fig. 4 A), no or very weak immunoreactivity for NNMT in the cytoplasm was observed in other OCCA specimens (Fig. 4 B). The association of cytoplasmic NNMT expression with clinicopathological characteristics of the 39 patients with OCCA is shown in Table 1 . Based on the correlation with the clinicopathological characteristics, NNMT expression showed no significant correlation with age, FIGO stage, tumor size, lymph node metastasis, distant metastasis, and optimal surgery. No deaths occurred in patients with lower levels of NNMT expression in the cytoplasm of tumor cells during the study period, whereas 18% (7 of 39 cases) of patients with higher levels of NNMT expression in the cytoplasm of tumor cells died. The log-rank test showed no significant difference between the two groups ( p = 0.155), but the hazard ratio (Mantel-Haenszel method) was 3.6, indicating a 3.6-fold higher mortality in the group with NNMT expression (Fig. 4 C). A chi-square test of likelihood ratios for the presence or absence of NNMT expression and whether the patient died or survived showed a trend toward more deaths in the high NNMT expression group than in the low NNMT expression group ( p = 0.0789). Patients with high NNMT immunoreactivity in the tumor stroma had significantly shorter progression-free survival than those with low NNMT immunoreactivity ( p = 0.0115, log-rank test) (data not shown). Table 1 Association of NNMT expression with clinicopathological characteristics of 39 ovarian clear cell adenocarcinomas. Patient characteristics n NNMT h (%) Pearson’s χ 2 P Total 39 32 (82.1) Age (years) < 60 (25) 22 (88.0) 1.673 0.196 ≥ 60 (14) 10 (71.4) Stage Ⅰ 27 22 (81.5) 3.188 0.364 Ⅱ 5 5 (100) Ⅲ 5 3 (60.0) Ⅳ 2 2 (100) Primary tumor size T1 28 22 (78.6) 1.331 0.514 T2 6 5 (83.3) T3 5 5 (100) Lymph node metastasis N0 35 30 (85.7) 3.109 0.078 N1 4 2 (50) Distant metastasis M0 37 30 (81.1) 0.461 0.497 M1 2 2 (100) Optimal surgery Optimal (< 1 cm) 32 26 (81.3) 0.077 0.780 Suboptimal ( ≧ 1 cm) 7 6 (85.7) NNMT h high NNMT expression. Knockdown of NNMT expression in OVMANA and RMG1 cells with NNMT siRNA reduced paclitaxel efficacy NNMT was knocked down using siRNA, and the decrease in NNMT protein expression was confirmed by western blotting (Fig. 5 D). Cell proliferation was examined using a WST-8 assay. The IC50 of OVTOKO cells with siRNA knockdown of NNMT was lower than the IC50 of cells treated with control siRNA (< 0.7-fold, p = 0.00275, student t -test) (Fig. 5 A). Additionally, The IC50 of RMG1 cells with siRNA knockdown of NNMT was lower than the IC50 of cells treated with control siRNA (< 0.44-fold, p = 0.00190, student t -test) (Fig. 5 B). Using ES-2 cells without NNMT expression, we examined whether cell proliferation differed between the NNMT and the control siRNA-treated groups. Cell proliferation was similar in both groups ( p = 0.984, student t -test) (Fig. 5 C). Overexpression of NNMT in ES-2 cells did not alter their sensitivity to paclitaxel ES-2 cells were used to create a stable cell line overexpressing NNMT, and the increase in NNMT protein was confirmed by western blotting (Supplementary Fig. 3B). The NNMT-overexpressing line of ES-2 cells showed no change in proliferative ability after paclitaxel treatment compared to the control vector (pCMV vector)-transfected line ( p = 0.360, student t -test) (Supplementary Fig. 3A). Discussion Ovarian cancer is the fifth most common cancer-related death in the USA, with 12,810 women dying from it annually [ 1 ]. OCCA is rare; however, patients with advanced or recurrent disease have poor survival outcomes. Owing to intrinsic chemotherapy resistance, OCCA shows reduced sensitivity to chemotherapy [ 2 ]. Treatment failure due to chemotherapy resistance in advanced or recurrent cancers is a significant dilemma in the treatment of OCCA. Therefore, the search for indicators that can predict the efficacy of chemotherapy may improve the prognosis of patients with OCCA. Several biomarkers in tumor tissues have been used to predict the efficacy of chemotherapeutic drugs in ovarian cancer research and clinical settings. Demethylation of HIN-1 reverses paclitaxel resistance in OCCA through the AKT-mTOR signaling pathway [ 7 ]. Everolimus, administered after 5-aza-2-deoxycytidine treatment, is a promising therapy for paclitaxel-resistant OCCA and targets AKT/mTOR pathway and EZH2 factor [ 11 ]. A global proteomic study has been performed in 192 cases of OCCA, and the analysis has allowed us to separate the lipid metabolic activity cluster and the coagulation cluster. Accumulation of fibrinogen subunits in tumors within coagulation clusters may reflect the risk of developing deep vein thrombosis. Of particular interest is the increased lipid metabolic processes characteristic of OCCA, where the pale cytoplasm contains fat droplets in addition to glycogen, which may act as an alternative fuel for tumor growth, and findings suggesting an aggressive response to oxidative damage have also been observed [ 12 ]. However, more reliable biomarkers are required to predict the efficacy of chemotherapeutic agents against all OCCA molecular subtypes during treatment. In this study, we identified higher NNMT expression in the OVTOKO and OVMANA cell lines that showed reduced paclitaxel sensitivity than ES-2 and KK cells. NNMT is predominantly expressed in the liver and catalyzes the N-methylation of nicotinamide, pyridines, and other structural analogs involved in the biotransformation and detoxification of several drugs and xenobiotic compounds. To the best of our knowledge, there have been no direct studies on NNMT expression in OCCA. Growing evidence has shown that NNMT is aberrantly expressed in several cancers and is a promising prognostic predictor in cancers such as gastric carcinoma, oral squamous cell carcinoma, ovarian cancer, hepatocellular carcinoma, and nasopharyngeal carcinoma [ 13 – 17 ]. NNMT expression reduces sensitivity to anticancer drugs in several cancer cell types. For example, NNMT expression reduced apoptosis induced by adriamycin or paclitaxel in breast cancer cells [ 18 ]. Further, NNMT reduces sensitivity to 5-fluorouracil in colorectal cancer cells [ 19 ]. Thus, the role of NNMT in various carcinomas has evolved from a mere metabolic function to being a driving force in diseases, including various cancers. However, despite growing evidence that NNMT is an effective therapeutic target, no cell-active inhibitors of this enzyme have been developed. Expression in cancer-associated fibroblasts (CAFs) in highly atypical serous carcinomas in ovarian cancer, NNMT expression in CAFs has resulted in depletion of S-adenosylmethionine and decreased histone methylation associated with extensive gene expression changes in the tumor stroma [ 20 ]. Here, we investigated the effects of NNMT on paclitaxel resistance in OCCA cells. The OVTOKO and RMG1 cell lines, which show high NNMT expression, and the ES-2 cell line, which shows no NNMT expression, were selected for this study. NNMT knockdown increased paclitaxel sensitivity in OVTOKO and RMG1 cells, but not in ES-2 cells. Conversely, NNMT overexpression in ES-2 cells did not alter paclitaxel sensitivity. These results indicate that altering endogenous NNMT levels may affect paclitaxel sensitivity. NNMT inhibitors [ 21 ] may be used in the future to increase drug sensitivity in paclitaxel-resistant cancers. Cytoplasmic expression of NNMT may worsen the prognosis of OCCA. In high-grade serous ovarian carcinoma, NNMT expression in the tumor stroma is involved in the differentiation of cancer-associated fibroblasts, suggesting a potential role in cancer progression in the stroma [ 20 , 22 ]. However, NNMT expression in OCCA tumor cells may be involved in the association between chemotherapy sensitivity and prognosis. NNMT expression in ovarian cancer cells is associated with poor prognosis [ 13 ]. Although there are no reports in ovarian cancer, one study showed that NNMT expression in breast cancer tumor cells enhances chemotherapy resistance [ 18 ]. In our study, NNMT showed potential to serve as a biomarker for the prediction of diagnostic and chemotherapeutic efficacy in OCCA. In OCCA with NNMT expression, suppression of intrinsic NNMT expression may increase paclitaxel sensitivity. These results indicated that NNMT may be a potential therapeutic target for OCCA. However, the exact mechanism by which NNMT regulates paclitaxel sensitivity requires further investigation. In summary, our study demonstrated for the first time that NNMT is overexpressed in OCCA and high NNMT expression levels may correlate with poor survival outcomes and an unfavorable therapeutic response in patients who received chemotherapy. Further, reduced NNMT expression enhanced the sensitivity of OCCA cells to paclitaxel-induced cell death. Taken together, these results suggest that NNMT is a promising new therapeutic target for OCCA. Declarations Ethics approval and consent to participate: Approval of the research protocol by the Institutional Ethics Committee of the Medical Faculty at Teikyo University (13-003-4,22 October 2020). Written informed consent was obtained from all the patients for the use of their samples and collection in research. Consent for publication: Not applicable. Availability of data and materials: Not applicable. Competing interests: The authors declare that they have no competing interests. Funding: This work was supported by a clinical research fund from the Sasaki Foundation (M.S.), a Grant-in-Aid for Scientific Research C (grant no. 17K08709 to R.K.) from the Ministry of Education, Science, and Culture, Japan, and by a Grant-in-Aid for Scientific Research C (grant no. 19K09834 to K.N.) from the Ministry of Education, Science, and Culture, Japan. Authors’ Contributions : RK and YM performed the experiments and bioinformatic analyses, and pathological analyses and wrote the manuscript. YS performed the pathological analyses. KN supervised the analyses, interpreted data, and wrote the manuscript. RK, MS, YS, YM, HU, KU, KH, YT, KT, CK, HN, TI, MH, HH, and KN contributed reagents, materials, experimental techniques, and provided the clinical data. All authors have read and approved the final manuscript. Acknowledgments : We thank Ms. Kozue Suzuki at the Sasaki Foundation Sasaki Institute and Mr. Masato Watanabe at the Department of Pathology, Teikyo University School of Medicine, for technical assistance. We thank Dr. Takashi Sasaki of the Sasaki Institute for funding this research. References Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA Cancer J Clin. 2022;72:7-33. Gadducci A, Multinu F, Cosio S, Carinelli S, Ghioni M, Aletti GD. 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Nicotinamide N-methyltransferase overexpression is associated with Akt phosphorylation and indicates worse prognosis in patients with nasopharyngeal carcinoma. Tumour Biol. 2013;34:3923-31. Wang Y, Zeng J, Wu W, et al. Nicotinamide N-methyltransferase enhances chemoresistance in breast cancer through SIRT1 protein stabilization. Breast Cancer Res. 2019;21:64 . Eckert MA, Coscia F, Chryplewicz A, et al. Proteomics reveals NNMT as a master metabolic regulator of cancer-associated fibroblasts. Nature. 2019;569:723-8. Xie X, Liu H, Wang Y, et al. Nicotinamide N-methyltransferase enhances resistance to 5-fluorouracil in colorectal cancer cells through inhibition of the ASK1-p38 MAPK pathway. Oncotarget. 2016;7:45837-48. Gao Y, Martin NI, van Haren MJ. Nicotinamide N-methyl transferase (NNMT): An emerging therapeutic target. Drug Discov Today. 2021;26:2699-706. Harmankaya İ, Akar S, Uğraş S, Güler AH, Ezveci H, Aydoğdu M, Çelik Ç. Nicotinamide N-methyltransferase overexpression may be associated with poor prognosis in ovarian cancer. J Obstet Gynaecol. 2021;41:248-53. Additional Declarations No competing interests reported. Supplementary Files NNMTcancermeS1.pptx SupplementaryFiguresLegend.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4249856","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":292524119,"identity":"55e5f091-65fd-43dc-809b-288e3de3f2fd","order_by":0,"name":"Ryoko Kikuchi-Koike","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYDACHsZmxgaDBDn79gYgz8CCeC3GBjwHQFokiNHCwMzYwJCQuEEiAcQlQot5z+FmwxkFaYzbJZ9f3fCjQIKBv707Aa8WmbONzYkbDHKYLWfnlN3sATpM4szZDXi1SPAzNh98YFDBxnA7J+0GD1CLgUQucVp4GG6eSbv5hygtvBCHSRjcYD92mzhbeA4CvW+QZiDZk8N2W8ZAgoewX3jSH0v2/Emu72c//uzmmz82cvztvfi1IAEeAzBJrHIQYH9AiupRMApGwSgYQQAAUrFHha7jNdgAAAAASUVORK5CYII=","orcid":"","institution":"Teikyo University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Ryoko","middleName":"","lastName":"Kikuchi-Koike","suffix":""},{"id":292524120,"identity":"cb31aaa0-dd60-4431-8aa5-97d37958932d","order_by":1,"name":"Masaru Sakamoto","email":"","orcid":"","institution":"Sasaki Foundation Kyoundo Hospital","correspondingAuthor":false,"prefix":"","firstName":"Masaru","middleName":"","lastName":"Sakamoto","suffix":""},{"id":292524121,"identity":"883c55e3-9c2b-42ab-89ae-3d53fbd9545b","order_by":2,"name":"Yuko Sasajima","email":"","orcid":"","institution":"Teikyo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuko","middleName":"","lastName":"Sasajima","suffix":""},{"id":292524122,"identity":"44460877-6334-447f-810c-1e81c6296e1d","order_by":3,"name":"Yuko Miyagawa","email":"","orcid":"","institution":"Teikyo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuko","middleName":"","lastName":"Miyagawa","suffix":""},{"id":292524123,"identity":"ea12b979-45b2-4073-93c9-ae30b842b3f3","order_by":4,"name":"Hiroshi Uozaki","email":"","orcid":"","institution":"Teikyo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Uozaki","suffix":""},{"id":292524124,"identity":"62b4e573-b2e8-4d38-bf8e-c57f1ddd30b0","order_by":5,"name":"Kenji Umayahara","email":"","orcid":"","institution":"Sasaki Foundation Kyoundo Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kenji","middleName":"","lastName":"Umayahara","suffix":""},{"id":292524125,"identity":"55b83002-4857-4806-82d6-bf6576d8c073","order_by":6,"name":"Kei Hashimoto","email":"","orcid":"","institution":"Teikyo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kei","middleName":"","lastName":"Hashimoto","suffix":""},{"id":292524126,"identity":"3960ed27-454c-4ab3-8dd4-20b49f2d538f","order_by":7,"name":"Yuko Takahashi","email":"","orcid":"","institution":"Teikyo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuko","middleName":"","lastName":"Takahashi","suffix":""},{"id":292524127,"identity":"26bca205-7649-4085-aabe-4ab6ac8d034d","order_by":8,"name":"Kazuki Takasaki","email":"","orcid":"","institution":"Teikyo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kazuki","middleName":"","lastName":"Takasaki","suffix":""},{"id":292524128,"identity":"17a0d47f-88c6-4359-9ad5-504778c58a5a","order_by":9,"name":"Chikara Kihira","email":"","orcid":"","institution":"Teikyo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chikara","middleName":"","lastName":"Kihira","suffix":""},{"id":292524129,"identity":"116825a1-e46d-4829-9d74-77a3195c8439","order_by":10,"name":"Haruka Nishida","email":"","orcid":"","institution":"Teikyo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Haruka","middleName":"","lastName":"Nishida","suffix":""},{"id":292524130,"identity":"7b5ce319-d4f1-48e7-aaee-26409d35a992","order_by":11,"name":"Takayuki Ichinose","email":"","orcid":"","institution":"Teikyo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Takayuki","middleName":"","lastName":"Ichinose","suffix":""},{"id":292524131,"identity":"5e465403-9f0d-439a-b6f8-dea19e8dc8fc","order_by":12,"name":"Mana Hirano","email":"","orcid":"","institution":"Teikyo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mana","middleName":"","lastName":"Hirano","suffix":""},{"id":292524132,"identity":"d8292b10-1359-4f40-97a0-bafbba3599ea","order_by":13,"name":"Haruko Hiraike","email":"","orcid":"","institution":"Teikyo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Haruko","middleName":"","lastName":"Hiraike","suffix":""},{"id":292524133,"identity":"3442a0f1-f8d5-4dbb-ba4e-208149ee6632","order_by":14,"name":"Kazunori Nagasaka","email":"","orcid":"","institution":"Teikyo University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kazunori","middleName":"","lastName":"Nagasaka","suffix":""}],"badges":[],"createdAt":"2024-04-11 02:44:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4249856/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4249856/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55323444,"identity":"2b515538-b614-4079-a051-918b63e8c024","added_by":"auto","created_at":"2024-04-25 16:44:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":148722,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition rate of the number of viable cells in the ovarian clear cell carcinoma (OCCA) cell lines, OVTOKO, OVMANA, ES-2, and KK assessed at indicated paclitaxel concentrations via the WST-8 assay. IC50 for OVTOKO and OVMANA was higher than IC50 for KK and ES-2.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-4249856/v1/ae2df3bfc5e413ac7fc70274.png"},{"id":55323447,"identity":"24431485-86e3-45ec-aa48-1ab3069ad1cc","added_by":"auto","created_at":"2024-04-25 16:44:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":459862,"visible":true,"origin":"","legend":"\u003cp\u003eA. Proteins extracted from OVTOKO, OVMANA, ES-2, and KK cells were separated using 2D-DIGE and the protein expression changes were analyzed using the PDQuest software. Image analysis using PDQuest software identified 5 protein spots with significantly higher or lower expression in OVTOKO and OVMANA than that in ES-2 and KK cells. B. A representative gel image of 2D-DIGE is shown. The protein from ES-2 and OVMANA cells were labeled with Cy3 (green) and Cy5 (red), respectively. The red protein spot contained NNMT and its expression was higher in OVMANA than in ES-2 cells.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-4249856/v1/6ffa673524585831d48e10a7.png"},{"id":55324727,"identity":"1aaa2735-23c1-4043-a91a-fb9cd7c8ec7e","added_by":"auto","created_at":"2024-04-25 16:52:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":115090,"visible":true,"origin":"","legend":"\u003cp\u003eA. The figure shows the proteins identified using the MASCOT peptide mass fingerprinting server in one of the 5 protein spots. MASCOT analysis showed a top score of 159 for nicotinamide N-methyltransferase (NNMT). B. Western blot analysis showed that NNMT was expressed in OVTOKO and OVMANA, but not in ES-2 and KK cells.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-4249856/v1/723446b663b642c1119c971e.png"},{"id":55323445,"identity":"283b81a9-25d9-4dc0-8e44-f8fc89b96b6b","added_by":"auto","created_at":"2024-04-25 16:44:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":547078,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis of NNMT expression in primary OCCA tumors. A and B, representative NNMT immunohistochemical staining of primary ovarian clear cell carcinoma cells. High (A) or almost no (B) expression of NNMT was observed in the cytoplasm of primary OCCA cells. Magnification, ×400. C, Kaplan–Meier curve showing the overall survival rates of patients with OCCA. There were no deaths in patients with OCCA, who showed lower NNMT levels in the cytoplasm.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-4249856/v1/40649090c6c2d88f846184dd.png"},{"id":55324728,"identity":"09af806b-d7f4-49ef-9a9f-c429c6e79424","added_by":"auto","created_at":"2024-04-25 16:52:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":190927,"visible":true,"origin":"","legend":"\u003cp\u003eWe investigated changes in paclitaxel sensitivity after NNMT siRNA-dependent knockdown of OVTOKO and RMG1 using the WST-8 assay. The graphs were created using the mean of the three experiments and SEM is shown as error bars. A. Paclitaxel IC50 in the NNMT siRNA-transfected OVTOKO cells is significantly lower than that of the control siRNA-transfected OVTOKO cells (\u0026lt;0.7-fold, \u003cem\u003ep\u003c/em\u003e = 0.00275, student \u003cem\u003et\u003c/em\u003e-test). B. Paclitaxel IC50 in the NNMT siRNA-transfected RMG1 cells is significantly lower than that of the control siRNA-transfected RMG1 cells (\u0026lt;0.44-fold, \u003cem\u003ep\u003c/em\u003e = 0.00190, student \u003cem\u003et\u003c/em\u003e-test). C. Paclitaxel IC50 in the NNMT siRNA-transfected ES-2 cells is not significantly lower than that of the control siRNA-transfected ES-2 cells (1.01-fold, \u003cem\u003ep \u003c/em\u003e= 0.984, student \u003cem\u003et\u003c/em\u003e-test). \u0026nbsp;D. Western blot analysis showed reduced expression of NNMT protein in OVTOKO and RMG1 cells in the NNMT siRNA-treated group compared to the control siRNA-treated group. No difference was observed between the two groups in ES-2 cells because of the absence of NNMT expression.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-4249856/v1/c7609577131d24e969b5ed82.png"},{"id":59724602,"identity":"e636e781-f913-4236-8ffe-da1211e45d75","added_by":"auto","created_at":"2024-07-05 10:29:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2746458,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4249856/v1/9b73d861-f171-4599-befd-d351887deac4.pdf"},{"id":55323451,"identity":"62d616f6-1845-4c6e-ab2a-533064593a4a","added_by":"auto","created_at":"2024-04-25 16:44:59","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3870012,"visible":true,"origin":"","legend":"","description":"","filename":"NNMTcancermeS1.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4249856/v1/0f51f2a3ae944f142d13c3e7.pptx"},{"id":55323448,"identity":"4f30eb0c-3744-483a-803a-9e37053d6f09","added_by":"auto","created_at":"2024-04-25 16:44:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13182,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresLegend.docx","url":"https://assets-eu.researchsquare.com/files/rs-4249856/v1/16097672974e009aeb2c0424.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nicotinamide N-methyltransferase enhances paclitaxel resistance in ovarian clear cell carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOvarian cancer is the fifth most common cause of cancer-related deaths in women after lung, breast, colorectal, and pancreatic cancers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Ovarian clear cell adenocarcinoma (OCCA) is a rare and distinct histological type of epithelial ovarian cancer (EOC). Women diagnosed with OCCA are usually younger and at an earlier stage of cancer than those with the common high-grade serous adenocarcinoma histology [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The overall prognosis of OCCA is good because most cases are identified during stage I of cancer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, advanced and recurrent disease is associated with a poor prognosis and resistance to standard treatment [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. While serous adenocarcinoma, the most common type of ovarian cancer, responds well to anticancer drug therapy, ovarian clear cell carcinoma is known to have an abysmal prognosis due to the low efficacy of anticancer drug therapy. Although there are many patients with this type of ovarian clear cell carcinoma in Asia, the number of patients with this type of cancer is relatively small in Europe and the United States, and no progress has been made in developing therapeutic agents. Patients with OCCA show a lower response rate to paclitaxel and carboplatin treatment regimens than the non-OCCA ovarian cancer group [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Improving the response rate of patients with OCCA to paclitaxel and carboplatin regimens may improve the prognosis of patients with OCCA. Aoki et al. reported that taxane-based chemotherapy was effective in patients with OCCA that were positive for beta-tubulin III [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Ho et al. reported that restoration of HIN1 expression reversed paclitaxel resistance in OCCA [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Yet, the molecules involved in the efficacy of taxane-based chemotherapy for OCCA have not been identified. A retrospective analysis of a multicenter, Phase 3, randomized, controlled trial (JGOG3017/GCIG) comparing TC (carboplatin and paclitaxel) and CPT-P (irinotecan and cisplatin) therapy in patients with recurrent or persistent OCCA found that patients with platinum-resistant relapse had a significantly shorter median post-progression survival (PPS) compared to those with platinum-sensitive relapse [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Combination therapy with gemcitabine, cisplatin, and bevacizumab (GP\u0026thinsp;+\u0026thinsp;Bev) has also been reported to be promising for advanced OCCA [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The identification of molecules involved in paclitaxel resistance in OCCA may provide an opportunity to improve the efficacy of paclitaxel and carboplatin regimens in OCCA. Therefore, we aimed to analyze the effect of paclitaxel treatment on the OCCA cell lines ES-2, KK, OVMANA, and OVTOKO, and the associated molecular changes using fluorescence-labeled two-dimensional differential gel electrophoresis (2D-DIGE).\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and primary tumor samples\u003c/h2\u003e \u003cp\u003eOVTOKO and OVMANA cell lines were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan), ES-2 from the American Type Culture Collection, and KK from the National Defense Medical College [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Primary tumor samples were obtained during surgery from 39 patients treated at Teikyo University Hospital in Tokyo, with written consent obtained from each patient after approval was approved by the Clinical Ethics Committee of the Medical Faculty at Teikyo University (approval number: 13-003-4 on 22 October 2020). The study adheres to the provisions specified in the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eProliferation assay\u003c/h2\u003e \u003cp\u003eThe effects of paclitaxel on cell proliferation were evaluated using a WST-8 assay (Dojindo, Kumamoto, Japan). The assay can be used to evaluate cell viability in cell proliferation assays. WST-8 is reduced by dehydrogenase activity in cells to produce a water-soluble yellow-colored formazan dye formed by NADH produced in the mitochondria. The amount of formazan dye produced is directly proportional to the number of living cells. The protocol is described briefly as follows. Cells were seeded in 24-well plates and pre-incubated at 37\u0026deg;C for 24 h to enable cell attachment. At 72 h after drug treatment, 50 \u0026micro;L of WST-8 solution was added to each well and incubated at 37\u0026deg;C for 0.5\u0026ndash;4 h (the reaction time was adjusted according to the cell line). The augmentation of enzyme activity increased the amount of formazan dye, which was quantified using a microplate reader (Bio-Rad, Tokyo, Japan) by measuring absorbance at 450\u0026ndash;550 nm. This procedure was repeated at least three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTissue immunohistochemistry and immunofluorescence\u003c/h2\u003e \u003cp\u003eFormalin-fixed paraffin-embedded (FFPE) tissue specimens were sliced at 4 \u0026micro;m thickness, deparaffinized in xylene, and rehydrated using graded ethanol solutions. After activation in citric acid buffer at 98\u0026deg;C for 40 min, the slides were stained with anti-NNMT (Santa Cruz, Tokyo, Japan G-4; 1:100) antibodies and processed using the EnVision FLEX Visualization System (Agilent Technologies). Samples were counterstained with Mayer\u0026rsquo;s hematoxylin (131\u0026ndash;09665, Fujifilm, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTissue microarray analysis\u003c/h2\u003e \u003cp\u003eWe analyzed NNMT expression using a tissue microarray (TMA) for 39 patients with ovarian clear cell adenocarcinoma who underwent surgery at Teikyo University Hospital between January 2003 and December 2012. Detailed information about the clinical characteristics was obtained after a retrospective review of the medical records. NNMT immunohistochemical reactivity was scored without knowledge of the clinical outcomes by two observers (R.K. and Y.S.). Each sample was scored based on the percentage of positive cells in each compartment (0, no staining; 1, \u0026lt;\u0026thinsp;30%; 2, 30\u0026ndash;50%; 3, \u0026ge;\u0026thinsp;50%). The staining intensity was similar across all samples. Expression was considered \u0026lsquo;low\u0026rsquo; if the cytoplasmic staining intensity was 0 or 1 and \u0026lsquo;high\u0026rsquo; if scores of 2 or 3 were obtained. The analysis was limited to patients with ovarian clear cell carcinoma (N\u0026thinsp;=\u0026thinsp;39). Kaplan\u0026ndash;Meier survival curves and log-rank test were used to compare the difference and overall survival were generated using BellCurve for Excel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence-labeled two-dimensional differential gel electrophoresis (2D-DIGE)\u003c/h2\u003e \u003cp\u003eProteins were labeled using the CyDye DIGE Fluor minimal dye (GE Healthcare), as per the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eBriefly, 50 mg of a sample mixture extracted from the ovarian clear cell cancer cell lines (OVTOKO, OVMANA, ES-2, and KK) was adjusted to pH 8.5 using 50 mM NaOH, and samples were labeled with 400 pmol Cy5 as a control or with 400 pmol Cy3. Fluorescence labeling was performed on ice in the dark for 30 min and the reaction was subsequently quenched with 1 mL of 10 mM lysine (Sigma-Aldrich, St. Louis, USA) for 10 min. Each preparation was treated with two sample buffers containing 7 M urea, 2 M thiourea, 4% CHAPS, 1% immobilized pH gradient (IPG)-buffer with a pH range of 4\u0026ndash;7, and 2% dithiothreitol (DTT) according to the manufacture\u0026rsquo;s recommendations. The final volume was adjusted to 260 mL using rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.5% IPG buffer with a pH range of 4\u0026ndash;7, and 0.2% DTT). The mixture of proteins labeled with Cy3 and Cy5 was applied to Immobiline DryStrips (pH 4\u0026ndash;7, 13 cm) (Cytiva, Tokyo, Japan) and focused on a PROTEAN i12 IEF system (Bio-Rad, Tokyo, Japan). Focused IPG strips were equilibrated and loaded onto 12.5% SDS-polyacrylamide gels (30% acrylamide, 1.5 M Tris-HCl pH 8.8, 10% SDS, 10% ammonium persulfate, and 10% TetraMethylEthyleneDiamine (TEMED)) using low-fluorescence glass plates on an SE 600 Ruby system (GE Healthcare). All electrophoresis procedures were performed in the dark. After SDS-PAGE, gels were scanned using a Pharos FX System (Bio-Rad) with appropriate excitation/emission wavelengths specific for Cy3 (532/605 nm) and Cy5 (635/695 nm). Scanned images were analyzed using the PDQuest Advanced Version 8.0 software (Bio-Rad).\u003c/p\u003e \u003cp\u003ePDQuest was used to identify spots with higher or lower protein expression in ES-2 and KK, and OVMANA and OVTOKO, respectively. Five spots were identified. Mass spectrometric analysis showed that NNMT was present in one of the spots. The spot containing NNMT had higher protein levels in OVMANA and OVTOKO than in KK and ES-2. For further confirmation, ES-2 and OVMANA, ES-2 and OVTOKO, KK and OVMANA, KK, and OVTOKO were labeled with Cy3, Cy5, Cy5, and Cy3, respectively. The protein expression level of the spot containing NNMT was higher in OVMANA than ES-2, OVTOKO than ES-2, OVMANA than KK, and OVTOKO than KK.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePeptide mass fingerprinting (PMF)\u003c/h2\u003e \u003cp\u003eAll chemicals used in this study were of analytical grade. The compounds 4-Sulfophenyl isothiocyanate, a-cyano-4-hydroxycinnamic acid (CHCA), sodium bicarbonate, and ammonium bicarbonate were purchased from Sigma (St. Louis, MO, USA).\u003c/p\u003e \u003cp\u003eFor protein identification by peptide mass fingerprinting, protein spots were excised, digested with trypsin (Promega, Madison, WI), mixed with α-cyano-4-hydroxycinnamic acid in 50% acetonitrile /0.1% TFA, and subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis (Microflex LRF 20, Bruker Daltonics, MA, USA), as described by Fernandez et al. (Electrophoresis 19:1036\u0026ndash;1045). Spectra were collected from 300 shots per spectrum over an \u003cem\u003em/z\u003c/em\u003e range of 600\u0026ndash;3000 and calibrated by two-point internal calibration using trypsin auto-digestion peaks (\u003cem\u003em/z\u003c/em\u003e 842.5099 and 2211.1046). The peak list was generated using Flex Analysis 3.0. The threshold used for peak-picking was as follows: 500 for the minimum resolution of monoisotopic mass and 5 for S/N. The search program MASCOT, developed by Matrixscience (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.matrixscience.com/\u003c/span\u003e\u003cspan address=\"http://www.matrixscience.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), was used for protein identification using peptide mass fingerprinting. The following parameters were used for the database search: trypsin as the cleaving enzyme, maximum of one missed cleavage, iodoacetamide (Cys) as a complete modification, oxidation (Met) as a partial modification, monoisotopic masses, and a mass tolerance of \u0026plusmn;\u0026thinsp;0.1 Da. The PMF acceptance criterion included the probability scores.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eEqual amounts of protein were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membranes were blocked, the primary antibodies were added, and the membranes were incubated with secondary antibodies. Signals were detected using an Image Quant LAS 4000 Mini instrument (GE Healthcare, Wauwatosa, WI, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003esiRNA\u003c/h2\u003e \u003cp\u003eWe transfected NNMT siRNA (sc-61213; Santa Cruz Biotechnology) into the OVMANA, RMG1, and ES-2 cell lines cultured in 6-well plates using Lipofectamine RNAi MAX Reagent (13778; Invitrogen, Tokyo, Japan) and Opti-MEM Reduced Serum Medium (31985070; Thermo Fisher Scientific, Tokyo, Japan). Control siRNA-A (sc-37007; Santa Cruz Biotechnology, Tokyo, Japan) was used as a negative control. The knockdown effect was tested by western blotting using an anti-NNMT antibody.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExpression plasmid transfection\u003c/h2\u003e \u003cp\u003eWe transfected the NNMT Expression Plasmid DNA (RC200641; OriGene Technologies, Inc., MD, USA) into the ES-2 cell line that was cultured in 6-well plates using Lipofectamine 2000 Transfection Reagent (11668019; Thermo Fisher Scientific) and Opti-MEM Reduced Serum Medium (31985070; Thermo Fisher Scientific Tokyo, Japan). The pCMV vector was used as a negative control for all experiments. The cells were incubated for 2 weeks in a medium containing G418 (1000 \u0026micro;g/mL; Thermo Fisher Scientific, Tokyo, Japan). We also established stable control clones expressing NNMT in these cell lines. Drug-resistant clones were further incubated in a medium with puromycin (58-58-2; Sigma-Aldrich, Tokyo, Japan) and tested for the knockdown effect by western blotting using the NNMT antibody.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe Student \u003cem\u003et\u003c/em\u003e-test was used to determine the statistical significance of the differences between the comparison groups in vitro. Error bars represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean. The relationship between NNMT expression and clinicopathological characteristics was analyzed using Pearson\u0026rsquo;s \u003cem\u003eχ2\u003c/em\u003e test. Data is equally distributed, and survival rates were calculated using the Kaplan\u0026ndash;Meier method and log-rank test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProliferation of OCCA cell lines was differentially inhibited by paclitaxel\u003c/h2\u003e \u003cp\u003eUsing the WST-8 assay, we showed that paclitaxel inhibited the proliferation of OVTOKO, OVMANA, ES-2, and KK cells in a dose-dependent manner. Paclitaxel inhibited the proliferation of OVTOKO and OVMANA cells more than that of ES-2 and KK (IC50\u0026thinsp;=\u0026thinsp;0.0826\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0054, 0.0703\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0092, 0.0127\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0027, 0.0047\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0002, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2D-DIGE analysis of OCCA cell lines and mass spectrometric analysis\u003c/h2\u003e \u003cp\u003eThe mixture of OVTOKO, OVMANA, KK, and ES-2 cellular proteins was labeled with Cy5 and used as an internal control. The OVTOKO, OVMANA, KK, and ES-2 proteins from individual cell lines were labeled with Cy3. The mixture of the proteins labeled with Cy3 and Cy5 was analyzed using four 2D-DIGE gels. A total of 130 protein spots were detected from the mixture of proteins from OVTOKO, OVMANA, KK, and ES-2 cells on each gel using PDQuest Advanced software, and all spots on each gel were matched. A total of 128 protein spots were detected, and the spots for OVTOKO, OVMANA, KK, and ES-2 cells, and all spots on each gel were matched using the PDQuest Advanced software.\u003c/p\u003e \u003cp\u003eWe detected four spots that showed greater intensity in OVTOKO and OVMANA than in KK and ES-2 cells, and one spot that showed greater intensity in KK and ES-2 than in OVTOKO and OVMANA cells by creating analysis sets using PDQuest Advanced software. The spots and quantitative graphs for each spot are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. The red bars in the quantitative graph show the number of spots from ES-2, KK, OVMANA, and OVTOKO cells (from left to right) and the green bars show the number of spots from the mixture of ES-2, KK, OVMANA, and OVTOKO cells on each gel (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The three spots were detected via Coomassie Brilliant Blue staining. The samples were next subjected to mass spectrometric analysis. In one of the three spots, an unnamed protein with a molecular weight of ~\u0026thinsp;36 KDa and a pI of 5.88 was detected in a MASCOT search with the top score of 161 (data not shown). In another one of the 3 spots with a molecular weight of ~\u0026thinsp;33 KDa and pI 4.99, we detected Mixture 1 (glyceraldehyde-3-phosphate dehydrogenase, chain A, annexin A2, and chain A, crystal structure of human Mu_crystallin at 2.6 \u0026Aring;) at the top score 350 (data not shown). In another one of the 3 spots with a molecular weight of ~\u0026thinsp;30 KDa and pI 5.56, we detected NNMT via a Mascot search with a top score of 159 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB shows the list of digested peptides derived from the spot, which was identified as NNMT in a MASCOT search. Supplementary Fig.\u0026nbsp;1B shows the NNMT protein sequence coverage of the digested peptides. We confirmed that the spot containing NNMT had higher protein levels in OVMANA than in ES-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), in OVMANA than in KK, in OVTOKO than in ES-2, and in OVTOKO than in KK cells (Supplementary Fig.\u0026nbsp;2A, B, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eValidation of the 2D-DIGE results via western blot analysis\u003c/h2\u003e \u003cp\u003eWe detected NNMT in OVTOKO and OVMMANA cells, but not in ES-2 and KK cells, using western blot analysis. Thus, the 2D-DIGE results were validated using western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAssociation between the expression level of NNMT and clinicopathologic characteristics in cases with primary tumors\u003c/h2\u003e \u003cp\u003eTo further clarify the clinical significance of NNMT levels in OCCA, the expression level of the protein in primary OCCA tissues was evaluated by immunohistochemistry using an NNMT-specific antibody. Although a few OCCA specimens frequently showed high levels of NNMT in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), no or very weak immunoreactivity for NNMT in the cytoplasm was observed in other OCCA specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The association of cytoplasmic NNMT expression with clinicopathological characteristics of the 39 patients with OCCA is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Based on the correlation with the clinicopathological characteristics, NNMT expression showed no significant correlation with age, FIGO stage, tumor size, lymph node metastasis, distant metastasis, and optimal surgery. No deaths occurred in patients with lower levels of NNMT expression in the cytoplasm of tumor cells during the study period, whereas 18% (7 of 39 cases) of patients with higher levels of NNMT expression in the cytoplasm of tumor cells died. The log-rank test showed no significant difference between the two groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.155), but the hazard ratio (Mantel-Haenszel method) was 3.6, indicating a 3.6-fold higher mortality in the group with NNMT expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). A chi-square test of likelihood ratios for the presence or absence of NNMT expression and whether the patient died or survived showed a trend toward more deaths in the high NNMT expression group than in the low NNMT expression group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0789). Patients with high NNMT immunoreactivity in the tumor stroma had significantly shorter progression-free survival than those with low NNMT immunoreactivity (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0115, log-rank test) (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAssociation of NNMT expression with clinicopathological characteristics of 39 ovarian clear cell adenocarcinomas.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient characteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003en\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNNMT\u003csup\u003eh\u003c/sup\u003e (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePearson\u0026rsquo;s χ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32 (82.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;60 (25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22 (88.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.673\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.196\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;60 (14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (71.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅠ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22 (81.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.364\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅡ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅢ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (60.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅣ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimary tumor size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22 (78.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.331\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.514\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (83.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymph node metastasis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 (85.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.078\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistant metastasis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 (81.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.461\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.497\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOptimal surgery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOptimal (\u0026lt;\u0026thinsp;1 cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26 (81.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.780\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSuboptimal (\u0026thinsp;≧\u0026thinsp;1 cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (85.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eNNMT\u003csup\u003eh\u003c/sup\u003e high NNMT expression.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of NNMT expression in OVMANA and RMG1 cells with NNMT siRNA reduced paclitaxel efficacy\u003c/h2\u003e \u003cp\u003eNNMT was knocked down using siRNA, and the decrease in NNMT protein expression was confirmed by western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Cell proliferation was examined using a WST-8 assay. The IC50 of OVTOKO cells with siRNA knockdown of NNMT was lower than the IC50 of cells treated with control siRNA (\u0026lt;\u0026thinsp;0.7-fold, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00275, student \u003cem\u003et\u003c/em\u003e-test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Additionally, The IC50 of RMG1 cells with siRNA knockdown of NNMT was lower than the IC50 of cells treated with control siRNA (\u0026lt;\u0026thinsp;0.44-fold, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00190, student \u003cem\u003et\u003c/em\u003e-test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Using ES-2 cells without NNMT expression, we examined whether cell proliferation differed between the NNMT and the control siRNA-treated groups. Cell proliferation was similar in both groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.984, student \u003cem\u003et\u003c/em\u003e-test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eOverexpression of NNMT in ES-2 cells did not alter their sensitivity to paclitaxel\u003c/h2\u003e \u003cp\u003eES-2 cells were used to create a stable cell line overexpressing NNMT, and the increase in NNMT protein was confirmed by western blotting (Supplementary Fig.\u0026nbsp;3B). The NNMT-overexpressing line of ES-2 cells showed no change in proliferative ability after paclitaxel treatment compared to the control vector (pCMV vector)-transfected line (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.360, student \u003cem\u003et\u003c/em\u003e-test) (Supplementary Fig.\u0026nbsp;3A).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOvarian cancer is the fifth most common cancer-related death in the USA, with 12,810 women dying from it annually [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. OCCA is rare; however, patients with advanced or recurrent disease have poor survival outcomes. Owing to intrinsic chemotherapy resistance, OCCA shows reduced sensitivity to chemotherapy [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Treatment failure due to chemotherapy resistance in advanced or recurrent cancers is a significant dilemma in the treatment of OCCA. Therefore, the search for indicators that can predict the efficacy of chemotherapy may improve the prognosis of patients with OCCA. Several biomarkers in tumor tissues have been used to predict the efficacy of chemotherapeutic drugs in ovarian cancer research and clinical settings. Demethylation of HIN-1 reverses paclitaxel resistance in OCCA through the AKT-mTOR signaling pathway [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Everolimus, administered after 5-aza-2-deoxycytidine treatment, is a promising therapy for paclitaxel-resistant OCCA and targets AKT/mTOR pathway and EZH2 factor [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. A global proteomic study has been performed in 192 cases of OCCA, and the analysis has allowed us to separate the lipid metabolic activity cluster and the coagulation cluster. Accumulation of fibrinogen subunits in tumors within coagulation clusters may reflect the risk of developing deep vein thrombosis. Of particular interest is the increased lipid metabolic processes characteristic of OCCA, where the pale cytoplasm contains fat droplets in addition to glycogen, which may act as an alternative fuel for tumor growth, and findings suggesting an aggressive response to oxidative damage have also been observed [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, more reliable biomarkers are required to predict the efficacy of chemotherapeutic agents against all OCCA molecular subtypes during treatment. In this study, we identified higher NNMT expression in the OVTOKO and OVMANA cell lines that showed reduced paclitaxel sensitivity than ES-2 and KK cells. NNMT is predominantly expressed in the liver and catalyzes the N-methylation of nicotinamide, pyridines, and other structural analogs involved in the biotransformation and detoxification of several drugs and xenobiotic compounds. To the best of our knowledge, there have been no direct studies on NNMT expression in OCCA. Growing evidence has shown that NNMT is aberrantly expressed in several cancers and is a promising prognostic predictor in cancers such as gastric carcinoma, oral squamous cell carcinoma, ovarian cancer, hepatocellular carcinoma, and nasopharyngeal carcinoma [\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. NNMT expression reduces sensitivity to anticancer drugs in several cancer cell types. For example, NNMT expression reduced apoptosis induced by adriamycin or paclitaxel in breast cancer cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Further, NNMT reduces sensitivity to 5-fluorouracil in colorectal cancer cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thus, the role of NNMT in various carcinomas has evolved from a mere metabolic function to being a driving force in diseases, including various cancers. However, despite growing evidence that NNMT is an effective therapeutic target, no cell-active inhibitors of this enzyme have been developed. Expression in cancer-associated fibroblasts (CAFs) in highly atypical serous carcinomas in ovarian cancer, NNMT expression in CAFs has resulted in depletion of S-adenosylmethionine and decreased histone methylation associated with extensive gene expression changes in the tumor stroma [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, we investigated the effects of NNMT on paclitaxel resistance in OCCA cells. The OVTOKO and RMG1 cell lines, which show high NNMT expression, and the ES-2 cell line, which shows no NNMT expression, were selected for this study. NNMT knockdown increased paclitaxel sensitivity in OVTOKO and RMG1 cells, but not in ES-2 cells. Conversely, NNMT overexpression in ES-2 cells did not alter paclitaxel sensitivity. These results indicate that altering endogenous NNMT levels may affect paclitaxel sensitivity. NNMT inhibitors [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] may be used in the future to increase drug sensitivity in paclitaxel-resistant cancers. Cytoplasmic expression of NNMT may worsen the prognosis of OCCA. In high-grade serous ovarian carcinoma, NNMT expression in the tumor stroma is involved in the differentiation of cancer-associated fibroblasts, suggesting a potential role in cancer progression in the stroma [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, NNMT expression in OCCA tumor cells may be involved in the association between chemotherapy sensitivity and prognosis. NNMT expression in ovarian cancer cells is associated with poor prognosis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Although there are no reports in ovarian cancer, one study showed that NNMT expression in breast cancer tumor cells enhances chemotherapy resistance [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In our study, NNMT showed potential to serve as a biomarker for the prediction of diagnostic and chemotherapeutic efficacy in OCCA. In OCCA with NNMT expression, suppression of intrinsic NNMT expression may increase paclitaxel sensitivity. These results indicated that NNMT may be a potential therapeutic target for OCCA. However, the exact mechanism by which NNMT regulates paclitaxel sensitivity requires further investigation.\u003c/p\u003e \u003cp\u003eIn summary, our study demonstrated for the first time that NNMT is overexpressed in OCCA and high NNMT expression levels may correlate with poor survival outcomes and an unfavorable therapeutic response in patients who received chemotherapy. Further, reduced NNMT expression enhanced the sensitivity of OCCA cells to paclitaxel-induced cell death. Taken together, these results suggest that NNMT is a promising new therapeutic target for OCCA.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eApproval of the research protocol\u0026nbsp;by\u0026nbsp;the\u0026nbsp;Institutional\u0026nbsp;Ethics Committee\u0026nbsp;of the Medical Faculty at Teikyo University (13-003-4,22 October 2020).\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from\u0026nbsp;all\u0026nbsp;the patients for the use of their samples and collection\u0026nbsp;in research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by a clinical research fund from the Sasaki Foundation (M.S.),\u0026nbsp;a Grant-in-Aid for Scientific Research C (grant no. 17K08709 to R.K.) from the Ministry of Education, Science, and Culture, Japan,\u0026nbsp;and\u0026nbsp;by a Grant-in-Aid for\u0026nbsp;Scientific\u0026nbsp;Research C (grant no. 19K09834 to K.N.) from the Ministry of Education, Science, and Culture, Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eRK\u0026nbsp;and\u0026nbsp;YM performed the experiments and bioinformatic analyses,\u0026nbsp;and pathological analyses and wrote the manuscript. YS performed the pathological analyses. KN supervised the analyses, interpreted data, and wrote the manuscript. RK, MS, YS, YM, HU, KU, KH, YT, KT, CK, HN, TI, MH, HH, and KN contributed reagents, materials, experimental techniques, and provided\u0026nbsp;the\u0026nbsp;clinical data. All authors\u0026nbsp;have\u0026nbsp;read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eWe thank Ms. Kozue Suzuki at the Sasaki Foundation Sasaki Institute and Mr. Masato Watanabe at the Department of Pathology, Teikyo University School of Medicine, for technical assistance. We thank Dr. Takashi Sasaki of the Sasaki Institute for funding this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cstrong\u003eSiegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA Cancer J Clin. 2022;72:7-33.\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eGadducci A, Multinu F, Cosio S, Carinelli S, Ghioni M, Aletti GD.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eClear cell carcinoma of the ovary: Epidemiology, pathological and biological features, treatment options and clinical outcomes. Gynecol Oncol. 2021;162:741-50.\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003eCeppi L, Grassi T, Galli F, et al.\u0026nbsp;Early-stage clear cell ovarian cancer compared to high-grade histological subtypes: An outcome exploratory analysis in two oncology centers. Gynecol Oncol.\u0026nbsp;2021;160:64-70.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003ePenny SM. Ovarian Cancer: An Overview. Radiol Technol. 2020;91:561-75.\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eSirichaisutdhikorn D, Suprasert P, Khunamornpong S. Clinical outcome of the ovarian clear cell carcinoma compared to other epithelial ovarian cancers when treated with paclitaxel and carboplatin. Asian Pac. J. Cancer Prev. 2009;10:1041-5.\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eAoki D, Oda Y, Hattori S, et al. Overexpression of class III beta-tubulin predicts good response to taxane-based chemotherapy in ovarian clear cell adenocarcinoma. Clin Cancer Res. 2009;15: 1473-80.\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eHo CM, Huang CJ, Huang SH, Chang SF, Cheng WF. Demethylation of HIN-1 reverses paclitaxel-resistance of ovarian clear cell carcinoma through the AKT-mTOR signaling pathway. BMC Cancer. 2015;15:789\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eKondo E, Tabata T, Suzuki N, et al.\u0026nbsp;The post-progression survival of patients with recurrent or persistent ovarian clear cell carcinoma: results from a randomized phase III study in JGOG3017/GCIG. J Gynecol Oncol.\u0026nbsp;2020;31:e94.\u003c/li\u003e\n \u003cli\u003eIto K, Nakagawa M, Shimokawa M, et al. Phase II study of gemcitabine, cisplatin, and bevacizumab for first recurrent and refractory ovarian clear cell carcinoma Kansai Clinical Oncology Group-G1601. Anticancer Drugs. 2022.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eSasa H, Ishii K, Hirata J, et al.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEstablishment and characterization of a CA125-producing human ovarian clear cell carcinoma cell line. Hum Cell. 1993;6:279-86.\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eHo CM, Lee FK, Huang SH, Cheng WF. Everolimus following 5-aza-2-deoxycytidine is a promising therapy in paclitaxel-resistant clear cell carcinoma of the ovary. Am J Cancer Res. 2018;8:56-69.\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003eJi JX, Cochrane DR, Negri GL, et al. The proteome of clear cell ovarian carcinoma. J Pathol.\u0026nbsp;2022;258:325-338.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eZhang L, Song M, Zhang F, et al.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAccumulation of nicotinamide N-methyltransferase (NNMT) in cancer-associated fibroblasts: A potential prognostic and predictive biomarker for gastric carcinoma. J Histochem Cytochem. 2021;69:165-76.\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eZhang W, Jing Y, Wang S, et al. Identification of biological functions and prognostic value of NNMT in oral squamous cell carcinoma. Biomolecules. 2022;12:1487.\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eHarmankaya İ, Akar S, Uğraş S, et al. Nicotinamide N-methyltransferase overexpression may be associated with poor prognosis in ovarian cancer. J Obstet Gynaecol. 2021;41:248-53.\u0026nbsp;\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eKim J, Hong SJ, Lim EK, et al.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eExpression of nicotinamide N-methyltransferase in hepatocellular carcinoma is associated with poor prognosis. J Exp Clin Cancer Res. 2009;28:20.\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eWin KT, Lee SW, Huang HY, et al. Nicotinamide N-methyltransferase overexpression is associated with Akt phosphorylation and indicates worse prognosis in patients with nasopharyngeal carcinoma. Tumour Biol. 2013;34:3923-31.\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eWang Y, Zeng J, Wu W, et al.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eNicotinamide N-methyltransferase enhances chemoresistance in breast cancer through SIRT1 protein stabilization. Breast Cancer Res. 2019;21:64\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eEckert MA, Coscia F, Chryplewicz A, et al.\u0026nbsp;Proteomics reveals NNMT as a master metabolic regulator of cancer-associated fibroblasts. Nature.\u0026nbsp;2019;569:723-8.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eXie X, Liu H, Wang Y, et al.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eNicotinamide N-methyltransferase enhances resistance to 5-fluorouracil in colorectal cancer cells through inhibition of the ASK1-p38 MAPK pathway. Oncotarget. 2016;7:45837-48.\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eGao Y, Martin NI, van Haren MJ.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eNicotinamide N-methyl transferase (NNMT): An emerging therapeutic target. Drug Discov Today. 2021;26:2699-706.\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003eHarmankaya İ, Akar S, Uğraş S, G\u0026uuml;ler AH, Ezveci H, Aydoğdu M, \u0026Ccedil;elik \u0026Ccedil;. Nicotinamide N-methyltransferase overexpression may be associated with poor prognosis in ovarian cancer. J Obstet Gynaecol. 2021;41:248-53.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ovarian clear cell adenocarcinoma, chemoresistance, paclitaxel, nicotinamide N-methyltransferase, 2D-DIGE","lastPublishedDoi":"10.21203/rs.3.rs-4249856/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4249856/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground. Growing evidence suggests that nicotinamide N-methyltransferase (NNMT), an S-adenosyl-L-methionine (SAM)-dependent cytosolic enzyme, plays an essential role in cancer progression. Recently, it was reported that NNMT is involved in methylation metabolism and tumorigenesis and is associated with poor prognosis in a number of cancers. It has also been reported that NNMT is overexpressed in the stroma of advanced high-grade serous carcinomas and may contribute to poor survival. The aim of this study was to identify novel biomarkers that predict resistance in paclitaxel-resistant advanced or recurrent ovarian clear cell carcinoma (OCCA) and to evaluate their clinicopathologic significance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethods. Four OCCA cell lines (ES-2, KK, OVMANA, and OVTOKO) were divided into paclitaxel high and low sensitivity groups by WST-8 assay and fluorescence-labeled two-dimensional electrophoresis (2D-DIGE) was performed. Protein spots with different expression intensities in each drug-sensitive group were analyzed by mass spectrometry to identify the proteins.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults. NNMT was detected as a protein molecule upregulated in the paclitaxel-resistant group, and knockdown by NNMT siRNA increased paclitaxel sensitivity in the NNMT-expressing ovarian clear cell carcinoma cell lines OVTOKO and RMG1. Furthermore, in analysis of clinical tissue samples, no deaths were observed in 7 patients with low NNMT expression in the cytoplasm of cancer cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConclusions. High NNMT expression in the cytoplasm of cancer cells is associated with low sensitivity to paclitaxel in OCCA and may have prognostic implications; knockdown of NNMT expression also reduced paclitaxel efficacy. Therefore, targeted therapies that reduce cytoplasmic NNMT expression levels may increase the sensitivity of OCCA to paclitaxel.\u003c/p\u003e","manuscriptTitle":"Nicotinamide N-methyltransferase enhances paclitaxel resistance in ovarian clear cell carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-25 16:44:54","doi":"10.21203/rs.3.rs-4249856/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"016c982a-a63a-4591-a2fe-94869d0b58cd","owner":[],"postedDate":"April 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-08T08:21:24+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-25 16:44:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4249856","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4249856","identity":"rs-4249856","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}