Higher Nicotinamide N-methyltransferase Expression Research Articles

NNMT switches the proangiogenic phenotype of cancer-associated fibroblasts via epigenetically regulating ETS2/VEGFA axis.

Cancer-associated fibroblasts (CAFs) are known to promote angiogenesis in oral squamous cell carcinoma (OSCC). However, the epigenetic mechanisms through which CAFs facilitate angiogenesis within the tumor microenvironment are still poorly characterized. Nicotinamide N'-methyltransferase (NNMT), a member of the N-methyltransferase family, was found to be a key molecule in the activation of CAFs. This study shows that NNMT in fibroblasts contributes to angiogenesis and tumor growth through an epigenetic reprogramming-ETS2-VEGFA signaling axis in OSCC. Single-cell RNA Sequencing (scRNA-seq) analysis suggests that NNMT is mainly highly expressed in fibroblasts of head and neck squamous cell carcinoma (HNSCC). Moreover, analysis of the TCGA database and multiple immunohistochemical staining of clinical samples also identified a positive correlation between NNMT and tumor angiogenesis. This research further employed an assembled organoid model and a fibroblast-endothelial cell co-culture model to authenticate the proangiogenic ability of NNMT. At the molecular level, high expression of NNMT in CAFs was found to promote ETS2 expression by regulating H3K27 methylation level through mediating methylation deposition. Furthermore, ETS2 was verified to be an activating transcription factor of VEGFA in this study. Collectively, our findings delineate an epigenetic molecular regulatory network of angiogenesis and provide a theoretical basis for exploring new targets and clinical strategy in OSCC.

Nicotinamide N-Methyl Transferase as a Predictive Marker of Tubular Fibrosis in CKD.

Chronic kidney disease (CKD) progression is complex. There are not standardized methods for predicting the prognosis of CKD. Nicotinamide N-methyltransferase (NNMT) has been shown to be associated with renal fibrosis. This study aimed to validate NNMT as a prognostic biomarker of progressive CKD. We explored the relationship between NNMT expression and CKD-related outcome variables using the NephroseqV5 and GEO databases. Additionally, a validation set of 37 CKD patients was enrolled to measure the correlation between NNMT expression levels and CKD outcomes. Furthermore, single-cell RNA sequencing data and the Human Protein Atlas were reanalyzed to investigate the expression specificity of NNMT in the kidney. Finally, to detect the status of NNMT expression with tubular fibrosis in vivo, we constructed a unilateral ureteral obstruction (UUO) mouse treated with an NNMT inhibitor. Analyzing the datasets showed that NNMT was expressed mainly in proximal tubule compartments. And patients with high NNMT expression levels had a significantly lower overall survival rate compared to those with low NNMT expression levels (P = 0.013). NNMT was independent of prognosis factors in the multivariate Cox regression model, and the AUCs for CKD progression at 1, 3, and 5 years were 0.849, 0.775, and 0.877, respectively. Pathway enrichment analysis indicated that NNMT regulates the biological processes of tubulointerstitial fibrosis (TIF). In the validation group, NNMT levels were significantly higher in the CKD group combined with interstitial fibrosis. In vivo, NNMT was a high expression in the UUO group, peaking at postoperative day 21. Treatment with an NNMT inhibitor improved renal tubular interstitial fibrosis, and expression levels of FN, α-SMA, VIM, and TGF-β1 were decreased compared with UUO (P < 0.05). NNMT was expressed mainly in tubular renal compartments, and associated with CKD prognosis. It holds potential as a diagnostic biomarker for tubular fibrosis in CKD.

Abstract 3697: Modulation of Nicotinamide N-Methyl Transferase (NNMT) expression in TNBC is associated with altered cell membrane cholesterol levels

Abstract Introduction: Cancer cells often develop a program of metabolic adaptability and epigenetic remodelling to promote gene expression changes causing cancerous cell proliferation and invasion. The metabolic-epigenetic axis as a an underlying mechanism of carcinogenesis could lead to new approaches to cancer treatment. Nicotinamide N-Methyl Transferase (NNMT) is a metabolic enzyme that catalyzes the transfer of a methyl group from S-Adenosyl methionine (SAM) to nicotinamide, thereby decreasing the overall methyl pools for RNA, histone and DNA methylation. High NNMT expression has been previously associated with a poor outcome in TNBC. Hypothesis: We hypothesized that high versus low NNMT activity and resulting oncogenic gene expression in TNBC may alter histone methylation is association with altered metabolic pathway activation. Methods: We used two TNBC cell lines models, MDA-MB 231 and SUM159, to study the association between high versus low NNMT activity and metabolic pathway activation. Surprisingly, we identified gene expression pathways suggestive of increased free cholesterol biosynthesis gene expression with low NNMT, and then used biochemical assays and filipin to measure free cholesterol in both total high and low NNMT TNBC cell lysates and relative cholesterol expression in the High vs Low NNMT cell membranes. Results: In vitro, NNMT knockdown resulted in increased cholesterol synthetic pathway enzyme expression in both TNBC cell lines. In vivo studies also showed that protein expression of cholesterol synthesis enzymes was also increased in NNMT-depleted MDA-MB 231 xenografted tumors. Interestingly, the increased cholesterol level upon NNMT depletion was mainly localized at the plasma membrane via Filipin III staining, suggesting a possible association with less cellular membrane fluidity and a decreased oncogenic phenotype. In ongoing experiments, we are evaluating whether changes in histone methylation in high vs low NNMT cells are associated with differential expression of cholesterol biosynthetic genes, and free cholesterol production. We hypothesize that NNMT might epigenetically decrease the methylation of histones regulating cholesterol production in TNBC. Citation Format: Woei-Yaw Chee, Deniz Nesli Dolcen, Lynda Bennett, Suzanne D. Conzen. Modulation of Nicotinamide N-Methyl Transferase (NNMT) expression in TNBC is associated with altered cell membrane cholesterol levels. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3697.

NNMT promotes the progression of intrahepatic cholangiocarcinoma by regulating aerobic glycolysis via the EGFR-STAT3 axis

Nicotinamide N-methyltransferase (NNMT), a member of the N-methyltransferase family, plays an important role in tumorigenesis. However, its expression and biological functions in intrahepatic cholangiocarcinoma (iCCA) remain to be established. In our study, we identified NNMT as an oncogene in iCCA and provided mechanistic insights into the roles of NNMT in iCCA progression. High NNMT expression in iCCA tissues was identified using western blotting and immunohistochemistry (IHC). We identified a significantly higher NNMT expression level in human iCCA tissues than that in adjacent normal tissues. Increased NNMT expression promoted iCCA cell proliferation and metastasis in vitro and in vivo. Mechanistically, NNMT inhibited the level of histone methylation in iCCA cells by consuming the methyl donor S-adenosyl methionine (SAM), thereby promoting the expression of epidermal growth factor receptor (EGFR). EGFR may activate the aerobic glycolysis pathway in iCCA cells by activating the STAT3 signaling pathway. In conclusion, we identified NNMT as an oncogene in iCCA and provided mechanistic insights into the roles of NNMT in iCCA progression.

Nicotinamide N-methyltransferase expression in squamous cell carcinoma of the vulva

The molecular pathways involved in the development of vulvar squamous cell carcinoma (SCC) cancer are not completely known. Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme associated with tumorigenesis and metastasis in a variety of cancers. Its role in vulvar cancer has not been studied, previously. Vulvar SCC, high and low grade squamous intraepithelial lesions (SILs) and benign squamous hyperplasia were analysed immunohistochemically. The mean staining score for vulvar SCC was significantly higher than the score for vulvar squamous hyperplasia (p<.001). The mean relapse-free survival for patients with low and high NNMT expression was 41.4 months (95% CI: 25.6–57.2) and 19.8 months (95% CI: 3.0–36.6), respectively (p=.035). The mean disease-specific survival for patients with low and high NNMT expression was 75.8 months (95% CI: 57.5–94.2) and 27.8 months (95% CI 12.2–43.4), respectively (p=.015). Although quite preliminary, this study showed that NNMT expression was elevated in vulvar SCC compared to benign and premalignant lesions. Additionally, elevated NNMT expression was associated with poor survival. Impact Statement What is already known on this subject? Nicotinamide N-methyltransferase (NNMT) is a methyltransferase, associated with tumour progression, spread and poor prognosis in a variety of cancers. Its upregulation can lead to DNA hypomethylation, which can in turn result in the activation of proto-oncogenes and deactivation of tumour suppressor genes. What do the results of this study add? Although quite preliminary, this study showed that NNMT expression was elevated in vulvar SCC compared to benign and premalignant lesions. Additionally, elevated NNMT expression was associated with poor survival. What are the implications of these findings for clinical practice and/or further research? NNMT has been regarded as a potential target of cancer therapy and its role in vulvar cancer has not been studied, previously. This is the first study to investigate the expression of NNMT in vulvar cancer and associate NNMT elevation with poor survival. NNMT can further be investigated as a possible target of vulvar cancer therapy.

Prognostic value of nicotinamide N-methyltransferase in human cancers: Evidence from a meta-analysis and database validation.

BackgroundPrevious studies indicated that dysregulated expression of nicotinamide N-methyltransferase (NNMT) contributed to the tumor progression and predicted poor prognosis in various cancers. However, there was no exact conclusion on account of the contradictory results across studies.MethodsThe relevant studies up to December 7, 2020 were searched in PubMed, Web of Science, and Embase. The association between NNMT expression and prognostic outcomes was explored, including overall survival (OS), disease-free survival (DFS), and clinicopathological features. The bioinformatics database was used to validate the findings.ResultsThirteen retrospective studies containing 2,591 patients with cancers were included in this analysis. High NNMT expression was significantly associated with shorter OS (hazard ratio [HR] = 2.01, 95% confidence interval [CI] = 1.42–2.86, and P < 0.01) and DFS (HR = 1.59, 95% CI = 1.23–2.05, and P < 0.01) compared to low NNMT expression in cancers. Compared to patients with low NNMT expression, patients with high NNMT expression tended to have worse tumor differentiation (P = 0.03), earlier lymph node metastasis (P = 0.01), earlier distant metastasis (P = 0.02), and more advanced clinical stage (P < 0.01).ConclusionHigh NNMT expression is an unfavorable factor of various cancers. NNMT is a promising indicator to predict the prognosis of various cancers and can serve as a potential therapeutic target in various cancers.

Nicotinamide N-methyltransferase is related to MELF pattern invasion in endometrioid carcinoma.

Grade 1 (G1) endometrioid carcinoma (EC) is relatively a good prognosis. However, in a minority of cases, G1 shows an aggressive histological pattern known as the microcystic, elongated, and fragmented (MELF) pattern. We previously reported that EC with high expression levels of S100A4 and serum deprivation‐response protein (SDPR) was related to MELF pattern invasion. However, the molecular features of the invasive front area of the MELF pattern have not been investigated. In this study, we searched for genes preferentially expressed in the invasive front area of EC with the MELF pattern using laser microdissection and RNA sequencing, and showed that nicotinamide N‐methyltransferase (NNMT) is related to MELF pattern invasiveness. Immunohistochemical analyses confirmed high NNMT expression in the invasive front area of the MELF pattern. Moreover, NNMT promoted migration, invasion, colony formation, epithelial–mesenchymal transition (EMT), and chemoresistance using EC cell lines. We speculate that depletion of NNMT promotes histone methylation and leads to tumor suppression because NNMT consumes S‐adenosyl methionine (SAM), which is an essential methylation cofactor. NNMT knockout cells showed enhanced expression of H3K9me2. RNA sequencing using NNMT knockout cell lines suggested that methylation of H3K9 leads to repression of the transcription of various oncogenic genes. Our findings demonstrate the possibility that NNMT inhibitors, which are expected to be used for the treatment of metabolic disorders, would be effective for the treatment of aggressive EC. This is the first report of gene analyses focusing on the morphological changes associated with MELF pattern invasion of EC.

Patients with low nicotinamide N-methyltransferase expression benefit significantly from bevacizumab treatment in ovarian cancer

BackgroundThe role of nicotinamide N-methyltransferase (NNMT) in ovarian cancer is still elusive. Our aim is to explore the expression of NNMT in ovarian cancer and to assess its association with patient prognosis and treatment response.MethodsWe first analyzed the differential expression of NNMT among fallopian tube epithelium, primary ovarian cancers, metastatic ovarian cancers, and recurrent ovarian cancers using Gene Expression Ominus (GEO) database (GSE10971, GSE30587, GSE44104 and TCGA datasets). Then, we assessed the association of NNMT expression with clinical and molecular parameters using CSIOVDB database and GSE28739 dataset. Next, we evaluate the association of NNMT expression with the prognosis of ovarian cancer patients in both GSE9891 dataset and TCGA dataset. Finally, GSE140082 dataset was used to explore the association of NNMT expression with bevacizumab response.ResultsNNMT expression was significantly elevated in lymphovascular space invasion (LVSI)-positive ovarian cancers compared with that in LVSI-negative ovarian cancers (TCGA dataset, P < 0.05), Moreover, increased expression of NNMT was associated with increased tumor stage, grade, and mesenchymal molecular subtype (CSIOVDB database). Survival analysis indicated that increased expression of NNMT was associated with a reduced OS in both GSE9891 dataset (HR: 2.28, 95%CI: 1.51–3.43, Log-rank P < 0.001) and TCGA dataset (HR: 1.55, 95%CI: 1.02–2.36, Log-rank P = 0.039). Multivariate analysis further confirmed the negative impact of NNMT expression on OS in ovarian cancer patients in those two datasets. Furthermore, the NNMT-related nomogram showed that NNMT shared a larger contribution to OS, compared with debulking status. More interestingly, bevacizumab conferred significant improvements in OS for patients with low NNMT expression (HR: 0.56, 95%CI: 0.31–0.99, Log-rank P = 0.049). In contrast, patients with high NNMT expression didn’t benefit from bevacizumab treatment significantly (HR: 0.85, 95%CI: 0.48–1.49, Log-rank P = 0.561). NNMT expression was positively correlated with the expression of genes, LDHA and PGAM1, involved in Warburg effect.ConclusionsIn conclusion, NNMT expression is associated with the aggressive behavior of ovarian cancer, correlates with a poor prognosis, and is predictive of sensitivity to bevacizumab treatment.

High Expression of Nicotinamide N-Methyltransferase in Patients with Sporadic Alzheimer\u2019s Disease

We have previously shown that the expression of nicotinamide N-methyltransferase (NNMT) is significantly increased in the brains of patients who have died of Parkinson’s disease (PD). In this study, we have compared the expression of NNMT in post-mortem medial temporal lobe, hippocampus and cerebellum of 10 Alzheimer’s disease (AD) and 9 non-disease control subjects using a combination of quantitative Western blotting, immunohistochemistry and dual-label confocal microscopy coupled with quantitative analysis of colocalisation. NNMT was detected as a single protein of 29 kDa in both AD and non-disease control brains, which was significantly increased in AD medial temporal lobe compared to non-disease controls (7.5-fold, P < 0.026). There was no significant difference in expression in the cerebellum (P = 0.91). NNMT expression in AD medial temporal lobe and hippocampus was present in cholinergic neurones with no glial localisation. Cell-type expression was identical in both non-disease control and AD tissues. These results are the first to show, in a proof-of-concept study using a small patient cohort, that NNMT protein expression is increased in the AD brain and is present in neurones which degenerate in AD. These results suggest that the elevation of NNMT may be a common feature of many neurodegenerative diseases. Confirmation of this overexpression using a larger AD patient cohort will drive the future development of NNMT-targetting therapeutics which may slow or stop the disease pathogenesis, in contrast to current therapies which solely address AD symptoms.

Overexpression of Nicotinamide N-methyltransferase mainly covers stroma of colorectal cancer and correlates with unfavorable survival by its product 1-MNA.

Background: Accumulating evidence indicates that Nicotinamide N-methyltransferase (NNMT) is abnormally expressed in tumor tissues of several cancers including colorectal cancer (CRC) and associated with cancer progression. However, the distribution characteristics and the clinical value of each part of NNMT expression in CRC are still not fully understood. The purpose of this study is to determine the distribution of NNMT expression and its association with survival in CRC.Methods: By using the cancer genome atlas (TCGA) and clinical proteomic tumor analysis consortium (CPTAC), we firstly analyzed the difference of gene and protein levels of NNMT between CRC and normal colorectal tissue. Then, NNMT protein expressions were detected in 18 intraepithelial neoplastic samples and 177 CRC tumor samples through immunohistochemistry in our study cohort. Furthermore, the relationship between NNMT expression and clinicopathological characteristics, overall survival (OS) and disease-free survival (DFS) of CRC patients were analyzed by Pearson χ2 test and log-rank test, respectively, in public datasets and our study cohort. Lastly, the function of NNMT and its product 1-methyl-nicotinamide (1-MNA) on migration and invasion in colorectal cancer cells was analyzed by wound healing assay and transwell assay.Results: We determined that higher NNMT expression in CRC tissues than normal tissues in both gene and protein level in TCGA and CPTAC datasets (all p < 0.05). In addition, the strong relationships of NNMT expression with stromal cells were found in the TCGA cohort. Fortunately, our cohort could validate that the expression of NNMT in tumor stroma cell was significantly higher than that in tumor cell (p < 0.0001), and both of them were significantly higher than that in adjacent normal tissue (ANT) (p < 0.0001 and p < 0.0001, respectively). Furthermore, the positive NNMT expression in tumor cell and stromal cell were associated with series of unfavorable clinical characteristics, including advanced TNM stage, lymph node metastasis, distant metastasis (all p < 0.05). Also, higher NNMT was associated with unfavorable survival both in our study and public datasets, including TCGA and two Gene Expression Omnibus (GEO) datasets (GSE33113 and GSE17538). Moreover, the functional experiments showed that stromal cells with high NNMT expression can secret 1-MAN to promote migration and invasion of CRC cells in vitro.Conclusions: In CRC, NNMT is overexpressed in tumor cells and stroma cells, and then mainly expressed in tumor stroma cells. Overexpression of NNMT in tumor cell and stroma cell both are associated with metastasis and unfavorable survival. Besides, stromal cells with high NNMT expression secrets 1-MAN to promote migration and invasion of CRC cells. Therefore, NNMT may be a potential prognostic indicator in CRC patients.

Hepatoblastomas exhibit marked NNMT downregulation driven by promoter DNA hypermethylation.

Hepatoblastomas exhibit the lowest mutational burden among pediatric tumors. We previously showed that epigenetic disruption is crucial for hepatoblastoma carcinogenesis. Our data revealed hypermethylation of nicotinamide N-methyltransferase, a highly expressed gene in adipocytes and hepatocytes. The expression pattern and the role of nicotinamide N-methyltransferase in pediatric liver tumors have not yet been explored, and this study aimed to evaluate the effect of nicotinamide N-methyltransferase hypermethylation in hepatoblastomas. We evaluated 45 hepatoblastomas and 26 non-tumoral liver samples. We examined in hepatoblastomas if the observed nicotinamide N-methyltransferase promoter hypermethylation could lead to dysregulation of expression by measuring mRNA and protein levels by real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blot assays. The potential impact of nicotinamide N-methyltransferase changes was evaluated on the metabolic profile by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy. Significant nicotinamide N-methyltransferase downregulation was revealed in hepatoblastomas, with two orders of magnitude lower nicotinamide N-methyltransferase expression in tumor samples and hepatoblastoma cell lines than in hepatocellular carcinoma cell lines. A specific TSS1500 CpG site (cg02094283) of nicotinamide N-methyltransferase was hypermethylated in tumors, with an inverse correlation between its methylation level and nicotinamide N-methyltransferase expression. A marked global reduction of the nicotinamide N-methyltransferase protein was validated in tumors, with strong correlation between gene and protein expression. Of note, higher nicotinamide N-methyltransferase expression was statistically associated with late hepatoblastoma diagnosis, a known clinical variable of worse prognosis. In addition, untargeted metabolomics analysis detected aberrant lipid metabolism in hepatoblastomas. Data presented here showed the first evidence that nicotinamide N-methyltransferase reduction occurs in hepatoblastomas, providing further support that the nicotinamide N-methyltransferase downregulation is a wide phenomenon in liver cancer. Furthermore, this study unraveled the role of DNA methylation in the regulation of nicotinamide N-methyltransferase expression in hepatoblastomas, in addition to evaluate the potential effect of nicotinamide N-methyltransferase reduction in the metabolism of these tumors. These preliminary findings also suggested that nicotinamide N-methyltransferase level may be a potential prognostic biomarker for hepatoblastoma.

Nicotinamide N‑methyltransferase induces the proliferation and invasion of squamous cell carcinoma cells.

Cutaneous squamous cell carcinoma (cSCC) is a common malignancy initiated by keratinocytes of the epidermis, which are able to invade the dermis and its periphery. Although most patients with cSCC present with curable localized tumors, recurrence, metastasis and mortality occasionally occur. In the present study, nicotinamide N-methyltransferase (NNMT) was identified as an upregulated protein in the SCC12 cell line, which has high invasive potential compared with the SCC13 cell line. The effects of NNMT knockdown on proliferation, migration and invasion were investigated using SCC cells. shRNA-mediated downregulation of NNMT expression levels inhibited the proliferation and density-dependent growth of SCC12 cells. In addition, the results of a cell motility assay showed that the migration and invasion of SCC cells were markedly decreased in NNMT-knockdown cells. The assessment of epithelial-mesenchymal transition (EMT)-associated gene expression using PCR array analysis revealed that high NNMT expression levels were accompanied by high expression levels of EMT-associated genes, and that NNMT knockdown effectively suppressed the expression of matrix metalloproteinase 9, osteopontin, versican core protein and zinc finger protein SNAI2 in SCC12 cells. These results revealed that the upregulation of NNMT induced cellular invasion via EMT-related gene expression in SCC cells.

Metabolic reprogramming of the stromal epigenome in ovarian cancer metastasis

High grade serous ovarian cancer (HGSOC) is characterized by early metastasis throughout the peritoneal cavity and a poor clinical outcome. During the metastatic process, ovarian cancer cells interact with diverse cell populations, including mesothelial cells, adipocytes, and cancer‐associated fibroblasts (CAFs). Ovarian cancer CAFs have well‐described roles in promoting the growth and progression of ovarian cancer by secreting pro‐proliferative cytokines and oncogenic extracellular matrix components (ECM). To better understand the proteomic changes that occur during metastasis in both tumor and stromal compartments, laser‐capture microdissection was coupled to label‐free, quantitative proteomics to independently characterize the proteomes of both tumor and stromal compartments in a cohort of HGSOC patients. We identified a conserved stromal proteomic signature associated with metastasis which included high expression of nicotinamide N‐methyltransferase (NNMT) in the metastatic stroma. NNMT is a metabolic enzyme that catalyzes the methylation of nicotinamide and consumes S‐adenosyl methionine (SAM), thus depleting cellular methylation potential and reducing methylation of DNA and proteins. NNMT regulated the expression of thousands of genes and was necessary and sufficient for the reprogramming of normal fibroblasts to CAFs. Stromal NNMT expression altered multiple metabolic pathways and led to a significant reduction in cellular SAM pools. Attenuated DNA methylation and histone H3 lysine 27 trimethylation (H3K27me3) were associated with global changes in gene expression. Using genetic and small‐molecule inhibitor strategies, we found that stromal NNMT activity promoted the progression and metastasis of ovarian cancer. This study provides support to the idea that stromal methyltransferase activities can be targeted to normalize the metastatic stroma and should be further explored as treatment targets for cancer.Support or Funding InformationThis work was supported by a Marsha Rivkin Foundation award (MAE), National Cancer Institute (NCI) grants CA111882 and CA211916 (EL), the Harris Family Foundation (MAE and SDY), the Ludwig Institute for Cancer Research (EL), NIH grant CA175399 and DP2GM128199 (REM), V Foundation for Cancer Research (V2016‐020 to REM), the Körber Foundation/Körber European Science Prize (MM), the Max‐Planck Society for the Advancement of Science (MM), the Novo Nordisk Foundation (Grant agreement NNF14CC0001 and NNF15CC0001; FC and MM), and University of Chicago Cancer Center Support Grant P30CA014599.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NNMT Regulates Cancer Immunity and Is a Potential Prognostic Biomarker in Gastric and Colorectal Cancers

Background: Nicotinamide N-methyltransferase (NNMT), a cytosolic enzyme that catalyzes the N-methylation of nicotinamide, has been shown to be critical for promoting cancer progression, but little is known its role in gastric and colorectal cancers. Methods: We explored the role of NNMT in predicting prognosis and immune infiltration of gastric and colorectal cancers. Data was obtained from RNAseq Atlas, Oncomine, tumor Immune Estimation Resource (TIMER) databases, Kaplan-Meier plotter, PrognoScan database and Gene Expression Profiling Interactive Analysis (GEPIA). Findings: NNMT is overexpressed in gastric and colorectal cancers compared to adjacent normal tissues. High NNMT expression was significantly associated with worse outcome in gastric and colorectal cancer patients. However, high expression of NNMT was highly predictive of effectual 5-fluorouracil (5-FU) treatment in HER2 negative gastric cancer patients. Furthermore, there were significant positive correlations between NNMT expression and immune cells infiltration in gastric and colorectal cancers. Conversely, NNMT did not exert any significant effect on prognosis and immune infiltration in lung cancer. Interpretation: NNMT could be a prognostic factor for predicting clinical outcomes and immune infiltration, as well as a molecular target for the treatment of gastric and colorectal cancers. Funding: This work was supported in part by funding from the National Natural Science Foundation of China (No. 81502598). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: Not required.

Prognostic Value of Nicotinamide N-Methyltransferase Expression in Patients With Solid Tumors: A Systematic Review and Meta-Analysis.

Background: Nicotinamide N-methyltransferase (NNMT) is an enzyme that catalyzes N-methylation of pyridine-containing compounds. NNMT is upregulated in many types of solid tumors, suggesting the potential for its use as a tumor biomarker. However, the prognostic value of NNMT in solid tumors is still unclear. We therefore conducted a meta-analysis to investigate the association between NNMT expression and survival in patients with solid tumors.Methods: We focused on patients with solid tumors, using high NNMT expression levels as the intervention and low NNMT expression levels as the comparison, according to Patient, Intervention, Comparison, and Outcome (PICO) guidelines. Electronic databases (up to June 7, 2018) were comprehensively searched to collect relevant cohort studies regarding the associations between NNMT expression levels and survival outcomes (overall survival [OS], disease-specific survival [DSS] including cancer-specific survival [CSS], and time to tumor progression [TTP] including disease-free survival [DFS], progression-free survival [PFS], and metastasis-free survival [MeFS]). Publication biases were also examined. All analyses were performed using STATA 12.0 software.Results: A total of 3340 patients with solid tumors from nine published studies were included. The combined hazard ratio (HR) identified high NNMT expression levels as a poor prognostic predictor of OS (HR = 1.67, 95% CI = 1.23–2.26). However, NNMT levels had no significant association with DSS (HR = 1.47, 95% CI = 0.95–2.28) and TTP (HR = 1.13, 95%CI = 0.39–3.25).Conclusion: High NNMT expression levels may be a poor prognostic biomarker for patients with solid tumors.

An Epigenetic Signature in Adipose Tissue Is Linked to Nicotinamide N-Methyltransferase Gene Expression.

The enzyme nicotinamide N-methyltransferase (NNMT) is a major methyltransferase in adipose tissue. We hypothesized an epigenetic signature in association with NNMT gene expression in adipose tissue. The global human methylome was analyzed in visceral adipose tissue (VAT) from morbidly obese patients using the Infinium Human Methylation 450 BeadChip array (discovery cohort: n=11). The findings were confirmed in two additional independent cohorts (cohort 1: n=60; BMI 20-60 kg m-2 and cohort 2: n=40; BMI > 40 kg m-2 ) and validated after weight loss (using microarray data). Among the genes associated with the largest methylation fold change were genes related to metabolic processes, proliferation, inflammation, and extracellular matrix remodeling, such as COL23A1, PLEC1, FBXO21, STEAP3, RGS12, IGDCC3, FOXK2, and ORAI2. In fact, the results showed 577 differentially methylated CpG sites (DMCpGs) associated with the NNMT expression levels, with low methylation levels paralleling high NNMT expression. The expression of FBXO21 and FOXK2 was specifically modified after weight loss concomitantly with a decrease in NNMT expression and inflammation-related genes. Interestingly, the adipose tissue NNMT gene expression correlated with markers of adipose tissue inflammation. The expression of NNMT in VAT is associated with a specific methylome signature involving genes linked to adipose tissue metabolic pathophysiology.

Nicotinamide N-methyltransferase: potential involvement in cutaneous malignant melanoma.

Nicotinamide N-methyltransferase (NNMT) is an enzyme that catalyzes the N-methylation of nicotinamide and pyridine compounds, participating in xenobiotic and drug metabolism. Data on literature have evidenced a possible role of NNMT in many solid cancers, but no data are currently available in cutaneous melanoma. Recent important advances have been achieved in the treatment of advanced melanoma with targeted therapy and immunotherapy. However, the identification of biomarkers that can be used for the detection of early stage disease as well as for monitoring the therapeutic response during treatment is of utmost importance. The aim of this study was to study the possible role of NNMT in melanoma. In the present study, we carried out immunohistochemical analyses to evaluate the expression of the enzyme NNMT in 34 melanomas and 34 nevi. Moreover, we explored the relationship between NNMT levels and the prognostic parameters of patients with melanoma. The results obtained showed significantly (P<0.0001) higher NNMT expression in melanoma compared with that detected in nevi. In addition, a significant (P<0.05) inverse relationship was found between enzyme levels and Breslow thickness, Clark level, the presence/number of mitoses, and ulceration. Taken together, these data seem to suggest that NNMT could represent a molecular biomarker for melanoma, thus highlighting its potential for both diagnosis and prognosis of this neoplasm.

A small molecule inhibitor of Nicotinamide N-methyltransferase for the treatment of metabolic disorders

Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme that catalyzes the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) onto the substrate, nicotinamide (NA) to form 1-methyl-nicotinamide (MNA). Higher NNMT expression and MNA concentrations have been associated with obesity and type-2 diabetes. Here we report a small molecule analog of NA, JBSNF-000088, that inhibits NNMT activity, reduces MNA levels and drives insulin sensitization, glucose modulation and body weight reduction in animal models of metabolic disease. In mice with high fat diet (HFD)-induced obesity, JBSNF-000088 treatment caused a reduction in body weight, improved insulin sensitivity and normalized glucose tolerance to the level of lean control mice. These effects were not seen in NNMT knockout mice on HFD, confirming specificity of JBSNF-000088. The compound also improved glucose handling in ob/ob and db/db mice albeit to a lesser extent and in the absence of weight loss. Co-crystal structure analysis revealed the presence of the N-methylated product of JBSNF-000088 bound to the NNMT protein. The N-methylated product was also detected in the plasma of mice treated with JBSNF-000088. Hence, JBSNF-000088 may act as a slow-turnover substrate analog, driving the observed metabolic benefits.

Abstract 5899: Compartment-resolved proteomics reveal NNMT as a master metabolic regulator of cancer associated fibroblasts

Abstract The poor outcome of ovarian cancer patients is due to late detection and the ability of ovarian cancer cells to metastasize quickly throughout the abdominal cavity. Recent research, including histopathological studies and experiments with mouse models, suggest that ovarian cancer may actually arise in the fallopian tube as serous tubal intraepithelial carcinomas (STIC). Lesions of the ovary would, therefore, represent metastases from the fallopian tube. To better understand the molecular events that occur in both the tumor and stromal compartment during ovarian cancer progression, we performed shotgun proteomics on laser-microdissected tumor and stromal compartments from anatomic sites representing the hypothetical progression series for ovarian cancer (STIC → fallopian tube → ovary → omentum). With an optimized sample preparation technique, we successfully quantified 4-5000 proteins per anatomic site with high reproducibility and with as few as several thousand microdissected cells derived from formalin-fixed and paraffin-embedded (FFPE) biobank specimens. Proteomics of the tumor compartments revealed high inter-patient heterogeneity, with no conserved protein signatures associated with invasion or metastasis. In strong contrast, a highly conserved molecular signature of stromal proteins associated with metastasis to the omentum was identified. In particular, nicotinamide N-methyl transferase (NNMT) was highly upregulated in the stroma of omental and peritoneal metastases. Functionally, NNMT was necessary and sufficient for multiple aspects of the cancer associated fibroblast (CAF) phenotype, including expression of CAF markers and secretion of cytokines and oncogenic extracellular matrix. NNMT expression was necessary for the ability of CAFs to support ovarian cancer cell migration, proliferation, adhesion, and in vivo growth. Mechanistically, high expression of NNMT in CAFs led to a depletion of S-adenosyl methionine (SAM) and a reduction in histone methylation associated with gene expression changes in the tumor stroma. This work supports the use of ultra-low input proteomics for compartment-resolved identification of candidate drivers of disease phenotypes and identifies NNMT as a central, metabolic regulator of CAF differentiation. Citation Format: Mark A. Eckert, Fabian Coscia, Shawn Pan, Samantha M. Tienda, Agnieszka A. Chryplewicz, Chun-Yi Chiang, Anthony Montag, S. Diane Yamada, Matthias Mann, Ernst R. Lengyel. Compartment-resolved proteomics reveal NNMT as a master metabolic regulator of cancer associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5899. doi:10.1158/1538-7445.AM2017-5899

Abstract 1693: Nicotinamide N-methyltransferase in clear cell renal cell carcinoma primary tumors and metastases

Abstract Background/Purpose: Nicotinamide N-methyltransferase (NNMT) is an enzyme involved in the biotransformation of many drugs and xenobiotic compounds. It is overexpressed in different cancers and has been shown to enhance the migratory and invasive activity of cancer cells. Additionally, NNMT is an important regulator of the methylation potential in cells by consumption of S-adenosyl methionine (SAM), an important substrate for methyltransferases. By this mechanism NNMT reduces methylation of proteins such as certain histones leading to epigenetic remodeling and induced expression of tumor-promoting genes (Ulanovskaya et al, Nat. Chem. Biol. 2013). In clear cell renal cell carcinoma (ccRCC), the dominant histological subtype of renal cell cancer, NNMT is expressed at high levels compared to normal kidney tissue and could represent a novel therapeutic target, especially for the treatment of metastatic disease which is marked by a five-year survival rate of below 10% with currently available therapies. Methods: We analyzed NNMT in a ccRCC patient cohort from The Cancer Genome Atlas (TCGA) concerning mRNA expression (n = 463), somatic variations (n = 410) and association with clinical parameters and patient survival. In an independent collection of ccRCC tissues (n = 64), corresponding normal kidney tissues and metastatic tissues from different organs (n = 129), NNMT mRNA and protein expression were quantified via Real-Time PCR and immunohistochemical staining of tissue microarrays, respectively. Results: In both cohorts, NNMT mRNA expression was significantly upregulated in tumor compared to non-tumor tissue. Somatic mutations and copy number alterations in the NNMT gene locus were observed in only a small number of patients. NNMT mRNA expression correlated weakly with tumor stage and grading in both cohorts, but not with the presence of lymph node or distant metastases. In Kaplan-Meier analyses high NNMT expression was associated with worse overall and cancer-specific survival. In ccRCC metastases from different organ sites, NNMT mRNA and protein expression were also induced compared to normal kidney tissue and high expression was again associated with worse patient survival in Kaplan-Meier analysis (HR = 2.15, Plog-rank = 0.05). Conclusion: NNMT mRNA and protein are highly expressed not only in primary tissue of ccRCC, but also in metastases derived from different organs, offering it as a potential drug-target for metastatic ccRCC. Although NNMT expression is a good prognostic indicator for patient survival, its molecular function in ccRCC tumorigenesis and metastasis formation has to be further evaluated. Supported by the Robert Bosch Foundation, Stuttgart, Germany and the ICEPHA Grant Tuebingen-Stuttgart, Germany Citation Format: Anna Reustle, Steffen Rausch, Stefan Winter, Florian Büttner, Stephan Kruck, Joerg Hennenlotter, Marcus Scharpf, Falko Fend, Jens Bedke, Arnulf Stenzl, Matthias Schwab, Elke Schaeffeler. Nicotinamide N-methyltransferase in clear cell renal cell carcinoma primary tumors and metastases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1693.