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Abstract Human hallmarks of sarcopenia include muscle weakness and a blunted response to exercise. Nicotinamide N-methyltransferase inhibitors (NNMTis) increase strength and promote the regenerative capacity of aged muscle, thus offering a promising treatment for sarcopenia. Since human hallmarks of sarcopenia are recapitulated in aged (24-month-old) mice, we treated mice from 22 to 24 months of age with NNMTi, intensive exercise, or a combination of both, and compared skeletal muscle adaptations, including grip strength, longitudinal running capacity, plantarflexor peak torque, fatigue, and muscle mass, fiber type, cross-sectional area, and intramyocellular lipid (IMCL) content. Exhaustive proteome and metabolome analyses were completed to identify the molecular mechanisms underlying the measured changes in skeletal muscle pathophysiology. Remarkably, NNMTi-treated aged sedentary mice showed ~ 40% greater grip strength than sedentary controls, while aged exercised mice only showed a 20% increase relative to controls. Importantly, the grip strength improvements resulting from NNMTi treatment and exercise were additive, with NNMTi-treated exercised mice developing a 60% increase in grip strength relative to sedentary controls. NNMTi treatment also promoted quantifiable improvements in IMCL content and, in combination with exercise, significantly increased gastrocnemius fiber CSA. Detailed skeletal muscle proteome and metabolome analyses revealed unique molecular mechanisms associated with NNMTi treatment and distinct molecular mechanisms and cellular processes arising from a combination of NNMTi and exercise relative to those given a single intervention. These studies suggest that NNMTi-based drugs, either alone or combined with exercise, will be beneficial in treating sarcopenia and a wide range of age-related myopathies.

Human hallmarks of sarcopenia include muscle weakness and a blunted response to exercise. Nicotinamide N-methyltransferase inhibitors (NNMTis) increase strength and promote the regenerative capacity of aged muscle, thus offering a promising treatment for sarcopenia. Since human hallmarks of sarcopenia are recapitulated in aged (24-month-old) mice, we treated mice from 22 to 24 months of age with NNMTi, intensive exercise, or a combination of both, and compared skeletal muscle adaptations, including grip strength, longitudinal running capacity, plantarflexor peak torque, fatigue, and muscle mass, fiber type, cross-sectional area, and intramyocellular lipid (IMCL) content. Exhaustive proteome and metabolome analyses were completed to identify the molecular mechanisms underlying the measured changes in skeletal muscle pathophysiology. Remarkably, NNMTi-treated aged sedentary mice showed ~ 40% greater grip strength than sedentary controls, while aged exercised mice only showed a 20% increase relative to controls. Importantly, the grip strength improvements resulting from NNMTi treatment and exercise were additive, with NNMTi-treated exercised mice developing a 60% increase in grip strength relative to sedentary controls. NNMTi treatment also promoted quantifiable improvements in IMCL content and, in combination with exercise, significantly increased gastrocnemius fiber CSA. Detailed skeletal muscle proteome and metabolome analyses revealed unique molecular mechanisms associated with NNMTi treatment and distinct molecular mechanisms and cellular processes arising from a combination of NNMTi and exercise relative to those given a single intervention. These studies suggest that NNMTi-based drugs, either alone or combined with exercise, will be beneficial in treating sarcopenia and a wide range of age-related myopathies. [ABSTRACT FROM AUTHOR]

Background/aim: Uterine leiomyosarcomas (uLMS) are extremely rare high-grade tumors with a poor prognosis. Their etiopathogenesis remains largely unknown. The uterus is the most frequent site for LMS. uLMS and uterine leiomyoma (uLM) must frequently be differentiated in patients with a uterine mass. Nicotinamide N-methyltransferase (NNMT), a cytoplasmic protein, is involved in the progression and spread of a variety of cancer types. The expression of NNMT in a mesenchymal malignancy was not examined previously. This study represents the first investigation into NNMT expression in uLMS, uLM and benign uterine myometrium and correlates NNMT overexpression with worse prognosis in uLMS. Materials and methods: The expression of NNMT was investigated by immunohistochemistry on formalin-fixed paraffin-embedded tissue of uLMS in 31 patients, uLM in seven patients and benign myometrial in 31 patients. Results: The expression of NNMT in uLMS was markedly higher than in uLM and normal myometrial tissue (p < 0.001). The expression of NNMT in early stage uLMS was lower than in advanced stage disease (p = 0.034). NNMT expression was an independent prognostic factor in predicting recurrence-free survival in uLMS (p = 0.037). Conclusion: NNMT can aid in the preoperative differentiation of uLMS and uLM. The consequences of NNMT overexpression, such as the activation and inactivation of oncoproteins and tumor suppressor proteins, respectively, as well as the enrichment of the cancer stem cell population, overlap with the major mechanisms responsible for poor prognosis in mesenchymal tumors. NNMT may be investigated further in the context of antitumor treatment in patients with mesenchymal malignancies. [ABSTRACT FROM AUTHOR]

Background: Osteosarcoma (OS) is a primary bone malignancy that mostly affects young people, characterized by high metastatic potential, and a marked chemoresistance that is responsible for disease relapse in most patients. Therefore, it is necessary to identify novel molecules to setup targeted strategies to improve the clinical outcome. The enzyme nicotinamide N‐methyltransferase (NNMT) catalyses the N‐methylation of nicotinamide and other analogs, playing a crucial role in the biotransformation of drugs and xenobiotics. NNMT overexpression was reported in a wide variety of cancers, and several studies demonstrated that is able to promote cell proliferation, migration and resistance to chemotherapy. The aim of this study was to explore the potential involvement of NNMT in OS. Methods: Immunohistochemical analyses have been performed to evaluate NNMT expression in selected OS and healthy bone tissue samples. Subsequently, OS cell lines have been transfected with vectors targeting NNMT mRNA (shRNAs) and the impact of this downregulation on migration, cell proliferation, and response to chemotherapeutic treatment was also analysed by wound healing, MTT, SRB and Trypan blue assays, respectively. Results: Results showed that OS samples display a significantly higher NNMT expression compared with healthy tissue. Preliminary results suggest that NNMT silencing in OS cell lines is associated to a decrease of cell proliferation and migration, as well as to enhanced sensitivity to chemotherapy. Data obtained showed that NNMT may represent an interesting marker for OS detection and a promising target for effective anti‐cancer therapy. [ABSTRACT FROM AUTHOR]

Abstract The identification of effective therapeutic targets plays a pivotal role in advancing cancer treatment outcomes. We employed a comprehensive pan-cancer analysis, complemented by experimental validation, to explore the potential of Nicotinamide N-methyltransferase (NNMT) as a promising therapeutic strategy for human cancers. By analyzing large-scale transcriptomic datasets across various cancer types, we consistently observed upregulated expression of NNMT. Furthermore, elevated NNMT expression correlated with inferior overall survival in multiple cancer cohorts, underscoring its significance as a prognostic biomarker. Additionally, we investigated the relationship between NNMT expression and the tumor immune microenvironment, which plays a crucial role in regulating anti-tumor immune responses. To confirm the malignant functions of NNMT in tumor cells, we conducted a series of cell-based experiments, revealing that NNMT promotes cancer cell proliferation and invasion, indicative of its oncogenic properties. The integration of computational analysis and experimental validation in our study firmly establishes NNMT as a potential therapeutic target for human cancers. Specifically, targeting NNMT holds promise for the development of innovative and effective cancer treatments. Further investigations into NNMT's role in cancer pathogenesis could potentially pave the way for groundbreaking advancements in cancer treatment.

The identification of effective therapeutic targets plays a pivotal role in advancing cancer treatment outcomes. We employed a comprehensive pan-cancer analysis, complemented by experimental validation, to explore the potential of Nicotinamide N-methyltransferase (NNMT) as a promising therapeutic strategy for human cancers. By analyzing large-scale transcriptomic datasets across various cancer types, we consistently observed upregulated expression of NNMT. Furthermore, elevated NNMT expression correlated with inferior overall survival in multiple cancer cohorts, underscoring its significance as a prognostic biomarker. Additionally, we investigated the relationship between NNMT expression and the tumor immune microenvironment, which plays a crucial role in regulating anti-tumor immune responses. To confirm the malignant functions of NNMT in tumor cells, we conducted a series of cell-based experiments, revealing that NNMT promotes cancer cell proliferation and invasion, indicative of its oncogenic properties. The integration of computational analysis and experimental validation in our study firmly establishes NNMT as a potential therapeutic target for human cancers. Specifically, targeting NNMT holds promise for the development of innovative and effective cancer treatments. Further investigations into NNMT's role in cancer pathogenesis could potentially pave the way for groundbreaking advancements in cancer treatment. [ABSTRACT FROM AUTHOR]

Cellular metabolism-induced epigenetic regulation is essential for the maintenance of cellular homeostasis. Nicotinamide N-methyltransferase (NNMT) is emerging as a key point of intersection between cellular metabolism and epigenetic regulation and has a central role in various physiological and pathological processes. NNMT catalyzes the methylation of nicotinamide (NAM) using the universal methyl donor S-adenosyl methionine (SAM) to yield S-adeno-syl-L-homocysteine (SAH) and N1-methylnicotinamide (MNAM), directly linking methylation balance with nicotinamide adenosine dinucleotide (NAD+) contents. NNMT acts on either the SAM-methylation balance or both NAD+ metabolism, depending on the tissue involved or pathological settings where metabolic demand is increased. Under physiological conditions, the liver act as an essential metabolic organ with abundant NNMT expression, while NNMT hepatic function is not mediated by its methyltransferase activity due to other major methyltransferases such as glycine N-methyltransferase (GNMT) in the liver. However, hepatic NNMT, as well as its metabolite is improperly regulated and linked to the worse pathological states in liver diseases, including alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), liver cirrhosis, and hepatocellular carcinoma (HCC), suggesting a potential role in the process of liver diseases. In this review, we summarize how NNMT regulates cell methylation balance and NAD metabolism, and extensively outline the current knowledge concerning the functions of NNMT in hepatic metabolism including glucose, lipid and energy, with a specific focus on the contribution of NNMT to the pathophysiology of liver-related diseases. NNMT is involved in the development and progression of liver diseases. Understanding the complex NNMT regulatory network and its effects on pathogenesis could provide new therapeutic strategies in the context of liver diseases.

Background: Chronic kidney disease (CKD) is a significant global health issue characterized by progressive loss of kidney function. Renal interstitial fibrosis (TIF) is a common feature of CKD, but current treatments are seldom effective in reversing TIF. Nicotinamide N-methyltransferase (NNMT) has been found to increase in kidneys with TIF, but its role in renal fibrosis is unclear. Methods: Using mice with unilateral ureteral obstruction (UUO) and cultured renal interstitial fibroblast cells (NRK-49F) stimulated with transforming growth factor-β1 (TGF-β1), we investigated the function of NNMT in vivo and in vitro. Results: We performed single-cell transcriptome sequencing (scRNA-seq) on the kidneys of mice and found that NNMT increased mainly in fibroblasts of UUO mice compared to sham mice. Additionally, NNMT was positively correlated with the expression of renal fibrosis-related genes after UUO injury. Knocking down NNMT expression reduced fibroblast activation and was accompanied by an increase in DNA methylation of p53 and a decrease in its phosphorylation. Conclusions: Our findings suggest that chronic kidney injury leads to an accumulation of NNMT, which might decrease p53 methylation, and increase the expression and activity of p53. We propose that NNMT promotes fibroblast activation and renal fibrosis, making NNMT a novel target for preventing and treating renal fibrosis. [ABSTRACT FROM AUTHOR]

High-grade serous carcinoma has a poor prognosis, owing primarily to its early dissemination throughout the abdominal cavity. Genomic and proteomic approaches have provided snapshots of the proteogenomics of ovarian cancer1,2, but a systematic examination of both the tumour and stromal compartments is critical in understanding ovarian cancer metastasis. Here we develop a label-free proteomic workflow to analyse as few as 5,000 formalin-fixed, paraffin-embedded cells microdissected from each compartment. The tumour proteome was stable during progression from in situ lesions to metastatic disease; however, the metastasis-associated stroma was characterized by a highly conserved proteomic signature, prominently including the methyltransferase nicotinamide N-methyltransferase (NNMT) and several of the proteins that it regulates. Stromal NNMT expression was necessary and sufficient for functional aspects of the cancer-associated fibroblast (CAF) phenotype, including the expression of CAF markers and the secretion of cytokines and oncogenic extracellular matrix. Stromal NNMT expression supported ovarian cancer migration, proliferation and in vivo growth and metastasis. Expression of NNMT in CAFs led to depletion of S-adenosyl methionine and reduction in histone methylation associated with widespread gene expression changes in the tumour stroma. This work supports the use of ultra-low-input proteomics to identify candidate drivers of disease phenotypes. NNMT is a central, metabolic regulator of CAF differentiation and cancer progression in the stroma that may be therapeutically targeted.

Previous 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.

Background: Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that catalyzes the methylation of nicotinamide (NAM) to generate 1-methyl nicotinamide (MNAM). Although previous studies have shown that NNMT is frequently dysregulated to promote the onset and progression of many malignancies, its expression profile, prognostic value and function in oral squamous cell carcinoma (OSCC) are still unknown. Methods: We used untargeted metabolomics based on mass spectrometry to analyze potential metabolite differences between tumors and matched adjacent normal tissues in 40 OSCC patients. Immunohistochemistry (IHC) was used to analyze the NNMT expression profile in OSCC, and the diagnostic and prognostic values of NNMT were evaluated. Next, qPCR and Western blot were used to compare the expression of NNMT in five OSCC cell lines. Stable transfected cell lines were constructed, and functional experiments were carried out to elucidate the effects of NNMT on the proliferation and migration of OSCC cells. Finally, gene set enrichment analysis (GSEA) was performed using The Cancer Genome Atlas (TCGA) data to investigate the potential functional mechanisms of NNMT in OSCC. Results: We found that the nicotinamide metabolic pathway was abnormally activated in OSCC tumor tissues compared with normal tissues. NNMT was expressed ubiquitously in tumor cells (TCs) and fibroblast-like cells (FLCs) but was absent in tumor-infiltrating lymphocytes (TILs). OSCC patients with highly expressed NNMT in TCs had higher risk of lymph node metastasis and showed a worse pattern of invasion (POI). Moreover, patients with highly expressed NNMT were also susceptible to postoperative recurrence. Highly expressed NNMT can independently predict shorter disease-free survival and recurrence-free survival. Functionally, we demonstrated that the ectopic expression of NNMT promoted OSCC tumor cell proliferation and migration in vitro. Conversely, silencing exerted significantly opposite effects in vitro. In addition, GSEA showed that highly expressed NNMT was mainly enriched in the epithelial–mesenchymal transformation (EMT) pathway, which displayed a significant positive correlation with the six classic EMT markers. Conclusions: Our study uncovered that NNMT may be a critical regulator of EMT in OSCC and may serve as a prognostic biomarker for OSCC patients. These findings might provide novel insights for future research in NNMT-targeted OSCC metastasis and recurrence therapy. [ABSTRACT FROM AUTHOR]

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. 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. [ABSTRACT FROM AUTHOR]

Chronic hepatitis B virus (HBV) infection can lead to fibrosis, liver cirrhosis, and primary hepatocellular carcinoma. Investigating host factors that regulate HBV replication helps to identify antiviral targets. In the current study, we identified Nicotinamide N-Methyltransferase gene (NNMT) as a novel factor that regulates HBV transcription. NNMT is up-regulated at both the mRNA and protein levels in HepG2.2.15 cells compared to HepG2 cells. Overexpression of NNMT reduces HBV replication in several cell models, while knockdown of NNMT enhances HBV DNA levels. Mechanistically, NNMT suppresses HBV DNA replication by inhibiting HBV RNA transcription. The region required for the inhibitory effect of NNMT was narrowed to nt 1672–1708 in enhancer II by luciferase assays. On the other hand, ChIP assays and EMSA results showed that NNMT does not bind to this region substantially, either directly or indirectly. Next, a collection of hepatic nuclear receptor transcription factors was screened to determine whether they were affected by NNMT overexpression. NR5A1, a positive regulator of HBV replication, decreased significantly after NNMT overexpression. Collectively, the findings of this study shed light on the regulation of HBV transcription. • Nicotinamide N-Methyltransferase (NNMT) was upregulated upon HBV replication. • NNMT suppressed HBV replication by inhibiting transcription. • The HBV DNA sequence essential for NNMT's inhibitory action was determined. • NNMT inhibited NR5A1 expression. [ABSTRACT FROM AUTHOR]

Glucose is considered the primary energy source for all cells, and some cancers are addicted to glucose. Here, we investigated the functional consequences of chronic glucose deprivation in serous ovarian cancer cells. We found that cells resistant to glucose starvation (glucose-restricted cells) demonstrated increased metabolic plasticity that was dependent on NNMT (Nicotinamide N-methyltransferase) expression. We further show that ZEB1 induced NNMT, rendered cells resistant to glucose deprivation and recapitulated metabolic adaptations and mesenchymal gene expression observed in glucose-restricted cells. NNMT depletion reversed metabolic plasticity in glucose-restricted cells and prevented de novo formation of glucose-restricted colonies. In addition to its role in glucose independence, we found that NNMT was required for other ZEB1-induced phenotypes, such as increased migration. NNMT protein levels were also elevated in metastatic and recurrent tumors compared to matched primary carcinomas, while normal ovary and fallopian tube tissue had no detectable NNMT expression. Our studies define a novel ZEB1/NNMT signaling axis, which elicits mesenchymal gene expression, as well as phenotypic and metabolic plasticity in ovarian cancer cells upon chronic glucose starvation. Understanding the causes of cancer cell plasticity is crucial for the development of therapeutic strategies to counter intratumoral heterogeneity, acquired drug resistance and recurrence in high-grade serous ovarian cancer (HGSC).

We present a case study of a 20-year-old male with an unknown neurodegenerative disease who was referred to the Undiagnosed Diseases Network Vanderbilt Medical Center site. A previous metabolic panel showed that the patient had a critical deficiency in nicotinamide intermediates that are generated during the biosynthesis of NAD(H). We followed up on these findings by evaluating the patient’s ability to metabolize nicotinamide. We performed a global metabolic profiling analysis of plasma samples that were collected: (1) under normal fed conditions (baseline), (2) after the patient had fasted, and (3) after he was challenged with a 500 mg nasogastric tube bolus of nicotinamide following the fast. Our findings showed that the patient’s nicotinamide N-methyltransferase (NNMT), a key enzyme in NAD(H) biosynthesis and methionine metabolism, was not functional under normal fed or fasting conditions but was restored in response to the nicotinamide challenge. Altered levels of metabolites situated downstream of NNMT and in neighboring biochemical pathways provided further evidence of a baseline defect in NNMT activity. To date, this is the only report of a critical defect in NNMT activity manifesting in adulthood and leading to neurodegenerative disease. Altogether, this study serves as an important reference in the rare disease literature and also demonstrates the utility of metabolomics as a diagnostic tool for uncharacterized metabolic diseases.

Immune response to SARS-CoV-2 and ensuing inflammation pose a huge challenge to the host's nicotinamide adenine dinucleotide (NAD+) metabolism. Humans depend on vitamin B3 for biosynthesis of NAD+, indispensable for many metabolic and NAD+-consuming signaling reactions. The balance between its utilization and resynthesis is vitally important. Many extra-pulmonary symptoms of COVID-19 strikingly resemble those of pellagra, vitamin B3 deficiency (e.g., diarrhoea, dermatitis, oral cavity and tongue manifestations, loss of smell and taste, mental confusion). In most developed countries, pellagra is successfully eradicated by vitamin B3 fortification programs. Thus, conceivably, it has not been suspected as a cause of COVID-19 symptoms. Here, the deregulation of the NAD+ metabolism in response to the SARS-CoV-2 infection is reviewed, with special emphasis on the differences in the NAD+ biosynthetic pathway's efficiency in conditions predisposing for the development of serious COVID-19. SARS-CoV-2 infection-induced NAD+ depletion and the elevated levels of its metabolites contribute to the development of a systemic disease. Acute liberation of nicotinamide (NAM) in antiviral NAD+-consuming reactions potentiates "NAM drain", cooperatively mediated by nicotinamide N-methyltransferase and aldehyde oxidase. "NAM drain" compromises the NAD+ salvage pathway's fail-safe function. The robustness of the host's NAD+ salvage pathway, prior to the SARS-CoV-2 infection, is an important determinant of COVID-19 severity and persistence of certain symptoms upon resolution of infection. [ABSTRACT FROM AUTHOR]

Cutaneous melanoma, Immunohistochemistry, Lymph node metastases, Nevi, Nicotinamide N-methyltransferase 1 Immunohistochemical expression of NNMT in dermal melanocytic nevi (a; the scale bar corresponds to 100 m), primary cutaneous melanomas (b; the scale bar corresponds to 50 m) and lymph node metastases from cutaneous melanoma (c; the scale bar corresponds to 30 m). Keywords: Cutaneous melanoma; Immunohistochemistry; Lymph node metastases; Nevi; Nicotinamide N-methyltransferase EN Cutaneous melanoma Immunohistochemistry Lymph node metastases Nevi Nicotinamide N-methyltransferase 480 482 3 01/06/23 20230101 NES 230101 Davide Sartini and Elisa Molinelli have contributed equally to this work. [Extracted from the article]

For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs).  Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development.

Background ‒ Previous 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. Methods ‒ The 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. Results ‒ Thirteen 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). Conclusion ‒ High 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. [ABSTRACT FROM AUTHOR]

Abstract Background Nicotinamide N-methyltransferase (NNMT) is highly expressed in several cancers and can regulate cell epigenetic status and various cell metabolism pathways, such as ATP synthesis and cellular stress response. We reported in our previous papers that NNMT overexpression inhibits the apoptosis and enhances the chemotherapy resistance of breast cancer cells. This study aims to investigate the effect of NNMT on autophagy induced by oxidative stress in breast cancer cells, which might provide a novel therapeutic strategy for breast cancer treatment. Methods NNMT and LC3B II protein levels in the two cell models (SK-BR-3 and MDA-MB-231) with NNMT overexpression or knockdown were detected by Western blotting and correlated with each other. Changes in cellular viability, intracellular reactive oxygen species (ROS) and ATP levels were assessed after H2O2 treatment. Then, autophagosomes were imaged by transmission electron microscopy, and LC3 puncta were examined by confocal microscopy and flow cytometry. The LC3B II level and AMPK-ULK1 pathway activity were both detected by Western blotting to determine the role of NNMT in the H2O2-induced autophagy. Results NNMT expression was negatively correlated with LC3B II expression in both cell models (SK-BR-3 and MDA-MB-231). Then, NNMT overexpression attenuated the autophagy induced by H2O2 in SK-BR-3 cells, whereas knockdown promoted autophagy induced by H2O2 in MDA-MB-231 cells. Furthermore, mechanistic studies showed that NNMT suppressed the ROS increase, ATP decrease and AMPK-ULK1 pathway activation, resulting in the inhibition of H2O2-induced autophagy in breast cancer cells. Conclusions We conclude that NNMT inhibits the autophagy induced by oxidative stress through the ROS-mediated AMPK-ULK1 pathway in breast cancer cells and may protect breast cancer cells against oxidative stress through autophagy suppression.

Melanoma accounts for less than 5% of all cutaneous neoplasms but is responsible for the greater part of skin cancer–related deaths. Therefore, the identification of molecules that could serve as the therapeutic target is urgent. This study focused on the enzyme nicotinamide N‐methyltransferase (NNMT). The effect of NNMT knockdown on cell proliferation and migration of A375 melanoma cells was evaluated by MTT and wound healing assays, respectively. Viability of A375 cells downregulating NNMT was also explored under treatment with dacarbazine, a chemotherapeutic drug approved for advanced melanoma treatment. The impact of enzyme knockdown on cell proliferation and chemosensitivity was also investigated in WM‐115 melanoma cells. Results obtained demonstrated that NNMT silencing led to a significant reduction of cell proliferation and migration of A375 cells. Moreover, enzyme downregulation was associated with an increase of melanoma cells sensitivity to treatment with dacarbazine. Analogous effects induced by enzyme knockdown on cell proliferation and chemosensitivity were also found in the WM‐115 cell line. Our data seem to demonstrate that NNMT could represent a promising molecular target for the effective treatment of this form of skin cancer. [ABSTRACT FROM AUTHOR]

The anti-proliferative effects of 5-methylquinolinium (5MQ) of nicotinamide N-methyltransferase (NNMT) have not been previously investigated on a cervical cancer cell line. NNMT is a metabolic enzyme that is correlated with tumour progression and metastasis. 5MQ is a small molecule inhibitor of NNMT. 0.1–500 μM of 5MQ was tested on the HeLa epithelial cervical cancer cell line. Cell viability was assessed with the MTT test. TWIST, ZEB1, SERPIN1, SIRT1, CD16, mRNA and various protein expression levels were analysed with Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Western Blotting, respectively. 5MQ significantly inhibited HeLa cell proliferation in a concentration and time-dependent manner. Increased cell shrinkage, loss of cellular adhesions and apoptotic bodies were observed in HeLa cells after 5MQ treatment. Following treatment with 5MQ, ZEB1, SIRT1, CD16 mRNA levels were increased while TWIST and SERPIN1 mRNA levels were reduced. Expressions of oncogenic proteins phospho-Akt and SIRT1 were decreased. 5MQ can effectively inhibit HeLa cell proliferation without apparently affecting HEK-293 cell proliferation. What is already known on this subject? NNMT is a cytosolic enzyme involved in tumour progression, metastasis and treatment resistance. It was overexpressed in many human malignancies. 5-amino-1-methylquinolinium (5MQ) is a novel small molecule inhibitor of NNMT that has shown promising results in the treatment of obesity and in senescent muscle regeneration. 5MQ has not been tested on the HeLa cervical cancer cell line, previously. What do the results of this study add? In this study, 5MQ was tested on the HeLa cervical cancer cell line for the first time and the molecular changes associated with 5MQ treatment were analysed. 5MQ demonstrated significant anti-proliferative activity on HeLa cells, which displayed morphological signs of apoptosis. Treatment of HeLa cells with 5MQ led to an increase in ZEB1, SIRT1 mRNA while TWIST mRNA was decreased. Phospho-Akt and Sirtuin1 protein expressions were decreased. What are the implications of these findings for clinical practice and/or further research? 5MQ can effectively inhibit HeLa cell proliferation without apparently affecting HEK-293 cell proliferation. 5MQ treatment was associated with a decrease in the expression of phospho-Akt and Sirtuin1 proteins, both of which have been reported to maintain tumour progression. 5MQ can further be investigated and modified for anti-cancer therapy. [ABSTRACT FROM AUTHOR]

BackgroundAberrant expression of Nicotinamide N-methyltransferase (NNMT) has been reported in pancreatic cancer. However, the role of NNMT in pancreatic cancer development remains elusive. Therefore, the present study was to investigate the impact of NNMT on pancreatic cancer cell proliferation, metastatic potential and survival under metabolic stress.MethodsPancreatic cancer cell line PANC-1 was transfected with NNMT expression plasmid or small interfering RNA of NNMT to overexpress or knockdown intracellular NNMT expression, respectively. Rate of cell proliferation was monitored. Transwell migration and matrigel invasion assays were conducted to assess cell migration and invasion capacity. Resistance to glucose deprivation, sensitivity to glycolytic inhibition, mitochondrial inhibtion and resistance to rapamycin were examined to evaluate cell survival under metabolic stress.ResultsNNMT silencing markedly reduced cell proliferation, whereas NNMT overexpression promoted cell growth moderately. Knocking down NNMT also significantly suppressed the migration and invasion capacities of PANC-1 cells. Conversely, NNMT upregulation enhanced cell migration and invasion capacities. In addition, NNMT knockdown cells were much less resistant to glucose deprivation and rapamycin as well as glycolytic inhibitor 2-deoxyglucose whereas NNMT-expressing cells showed opposite effects although the effects were not so striking.ConclusionsThese data sugguest that NNMT plays an important role in PANC-1 cell proliferation, metastatic potential and survival under metabolic stress.

Background: Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that catalyzes the methylation of nicotinamide (NAM) to generate 1-methyl nicotinamide (MNAM). Although previous studies have shown that NNMT is frequently dysregulated to promote the onset and progression of many malignancies, its expression profile, prognostic value and function in oral squamous cell carcinoma (OSCC) are still unknown. Methods: We used untargeted metabolomics based on mass spectrometry to analyze potential metabolite differences between tumors and matched adjacent normal tissues in 40 OSCC patients. Immunohistochemistry (IHC) was used to analyze the NNMT expression profile in OSCC, and the diagnostic and prognostic values of NNMT were evaluated. Next, qPCR and Western blot were used to compare the expression of NNMT in five OSCC cell lines. Stable transfected cell lines were constructed, and functional experiments were carried out to elucidate the effects of NNMT on the proliferation and migration of OSCC cells. Finally, gene set enrichment analysis (GSEA) was performed using The Cancer Genome Atlas (TCGA) data to investigate the potential functional mechanisms of NNMT in OSCC. Results: We found that the nicotinamide metabolic pathway was abnormally activated in OSCC tumor tissues compared with normal tissues. NNMT was expressed ubiquitously in tumor cells (TCs) and fibroblast-like cells (FLCs) but was absent in tumor-infiltrating lymphocytes (TILs). OSCC patients with highly expressed NNMT in TCs had higher risk of lymph node metastasis and showed a worse pattern of invasion (POI). Moreover, patients with highly expressed NNMT were also susceptible to postoperative recurrence. Highly expressed NNMT can independently predict shorter disease-free survival and recurrence-free survival. Functionally, we demonstrated that the ectopic expression of NNMT promoted OSCC tumor cell proliferation and migration in vitro. Conversely, silencing exerted significantly opposite effects in vitro. In addition, GSEA showed that highly expressed NNMT was mainly enriched in the epithelial–mesenchymal transformation (EMT) pathway, which displayed a significant positive correlation with the six classic EMT markers. Conclusions: Our study uncovered that NNMT may be a critical regulator of EMT in OSCC and may serve as a prognostic biomarker for OSCC patients. These findings might provide novel insights for future research in NNMT-targeted OSCC metastasis and recurrence therapy.

Statins belong to the most often prescribed medications, which efficiently normalise hyperlipidaemia and prevent cardiovascular complications in obese and diabetic patients. However, beside expected therapeutic results based on the inhibition of 3‐hydroxyl‐3‐methylglutaryl‐CoA reductase, these drugs exert multiple side effects of poorly understood characteristic. In this study, side effects of pravastatin and atorvastatin on EA.hy926 endothelial cell line were investigated. It was found that both statins activate proinflammatory response, elevate nitric oxide and reactive oxygen species (ROS) generation and stimulate antioxidative response in these cells. Moreover, only slight stimulation of the mitochondrial biogenesis and significant changes in the mitochondrial network organisation have been noted. Although biochemical bases behind these effects are not clear, they may partially be explained as an elevation of AMP‐activated protein kinase (AMPK) activity and an increased activating phosphorylation of sirtuin 1 (Sirt1), which were observed in statins‐treated cells. In addition, both statins increased nicotinamide N‐methyltransferase (NNMT) protein level that may explain a reduced fraction of methylated histone H3. Interestingly, a substantial reduction of the total level of histone H3 in cells treated with pravastatin but not atorvastatin was also observed. These results indicate a potential additional biochemical target for statins related to reduced histone H3 methylation due to increased NNMT protein level. Thus, NNMT may directly modify gene activity. Beside expected therapeutic results, statins exert multiple side effects of poorly understood characteristic. In our study, side effects of pravastatin and atorvastatin on EA.hy926 endothelial cell line were investigated. It was found that both statins activate proinflammatory response, elevate nitric oxide and ROS generation, stimulate antioxidative response and cause significant changes in the mitochondrial network organisation. Moreover, both statins increased nicotinamide N‐methyltransferase (NNMT) protein level, which may influence histone H3 methylation, thus directly modifying gene activity. [ABSTRACT FROM AUTHOR]

The cell metabolome comprises abundant information that may be predictive of cell functions in response to epigenetic or genetic changes at different stages of cell proliferation and metastasis. An unbiased ultra-performance liquid chromatography-mass spectrometry-based metabolomics study revealed a significantly altered metabolome for human pancreatic carcinoma PANC-1 cells with gain-of-function non-coding microRNA-1291 (miR-1291), which led to a lower migration and invasion capacity as well as suppressed tumorigenesis in a xenograft tumor mouse model. A number of metabolites, including N-methylnicotinamide, involved in nicotinamide metabolism, and l-carnitine, isobutyryl-carnitine and isovaleryl-carnitine, involved in fatty acid metabolism, were elevated in miR-1291-expressing PANC-1. Notably, N-methylnicotinamide was elevated to the greatest extent, and this was associated with a sharp increase in nicotinamide N-methyltransferase (NNMT) mRNA level in miR-1291-expressing PANC-1 cells. In addition, expression of NNMT mRNA was inversely correlated with pancreatic tumor size in the xenograft mouse model. These results indicate that miR-1291-altered PANC-1 cell function is associated with the increase in N-methylnicotinamide level and NNMT expression, and in turn NNMT may be indicative of the extent of pancreatic carcinogenesis.

In obesity and type 2 diabetes, Glut4 glucose transporter expression is decreased selectively in adipocytes. Adipose-specific knockout or overexpression of Glut4 alters systemic insulin sensitivity. Here we show, using DNA array analyses, that nicotinamide N-methyltransferase (Nnmt) is the most strongly reciprocally regulated gene when comparing gene expression in white adipose tissue (WAT) from adipose-specific Glut4-knockout or adipose-specific Glut4-overexpressing mice with their respective controls. NNMT methylates nicotinamide (vitamin B3) using S-adenosylmethionine (SAM) as a methyl donor. Nicotinamide is a precursor of NAD(+), an important cofactor linking cellular redox states with energy metabolism. SAM provides propylamine for polyamine biosynthesis and donates a methyl group for histone methylation. Polyamine flux including synthesis, catabolism and excretion, is controlled by the rate-limiting enzymes ornithine decarboxylase (ODC) and spermidine-spermine N(1)-acetyltransferase (SSAT; encoded by Sat1) and by polyamine oxidase (PAO), and has a major role in energy metabolism. We report that NNMT expression is increased in WAT and liver of obese and diabetic mice. Nnmt knockdown in WAT and liver protects against diet-induced obesity by augmenting cellular energy expenditure. NNMT inhibition increases adipose SAM and NAD(+) levels and upregulates ODC and SSAT activity as well as expression, owing to the effects of NNMT on histone H3 lysine 4 methylation in adipose tissue. Direct evidence for increased polyamine flux resulting from NNMT inhibition includes elevated urinary excretion and adipocyte secretion of diacetylspermine, a product of polyamine metabolism. NNMT inhibition in adipocytes increases oxygen consumption in an ODC-, SSAT- and PAO-dependent manner. Thus, NNMT is a novel regulator of histone methylation, polyamine flux and NAD(+)-dependent SIRT1 signalling, and is a unique and attractive target for treating obesity and type 2 diabetes.

Gastrointestinal (GI) neoplasms include esophageal, gastric, colorectal, hepatic, and pancreatic cancers. They are characterized by asymptomatic behavior, being responsible for diagnostic delay. Substantial refractoriness to chemo- and radiotherapy, exhibited by late-stage tumors, contribute to determine poor patient outcome. Therefore, it is of outmost importance to identify new molecular targets for the development of effective therapeutic strategies. In this study, we focused on the enzyme nicotinamide N-methyltransferase (NNMT), which catalyzes the N-methylation reaction of nicotinamide and whose overexpression has been reported in numerous neoplasms, including GI cancers. The aim of this review was to report data illustrating NNMT involvement in these tumors, highlighting its contribution to tumor cell phenotype. Cited works clearly demonstrate the interesting potential use of enzyme level determination for both diagnostic and prognostic purposes. NNMT was also found to positively affect cell viability, proliferation, migration, and invasiveness, contributing to sustain in vitro and in vivo tumor growth and metastatic spread. Moreover, enzyme upregulation featuring tumor cells was significantly associated with enhancement of resistance to treatment with chemotherapeutic drugs. Taken together, these results strongly suggest the possibility to target NNMT for setup of molecular-based strategies to effectively treat GI cancers.

Background: The abundance of energy metabolites is intimately interconnected with the activity of chromatin-modifying enzymes in order to guarantee the finely tuned modulation of gene expression in response to cellular energetic status. Metabolism-induced epigenetic gene regulation is a key molecular axis for the maintenance of cellular homeostasis, and its deregulation is associated with several pathological conditions. Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that catalyzes the methylation of nicotinamide (NAM) using the universal methyl donor S-adenosyl methionine (SAM), directly linking one-carbon metabolism with a cell's methylation balance and nicotinamide adenine dinucleotide (NAD+) levels. NNMT expression and activity are regulated in a tissue-specific-manner, and the protein can act either physiologically or pathologically depending on its distribution. While NNMT exerts a beneficial effect by regulating lipid parameters in the liver, its expression in adipose tissue correlates with obesity and insulin resistance. NNMT upregulation has been observed in a variety of cancers, and increased NNMT expression has been associated with tumor progression, metastasis and worse clinical outcomes. Accordingly, NNMT represents an appealing druggable target for metabolic disorders as well as oncological and other diseases in which the protein is improperly activated. Scope of review: This review examines emerging findings concerning the complex NNMT regulatory network and the role of NNMT in both NAD metabolism and cell methylation balance. We extensively describe recent findings concerning the physiological and pathological regulation of NNMT with a specific focus on the function of NNMT in obesity, insulin resistance and other associated metabolic disorders along with its well-accepted role as a cancer-associated metabolic enzyme. Advances in strategies targeting NNMT pathways are also reported, together with current limitations of NNMT inhibitor drugs in clinical use. Major conclusions: NNMT is emerging as a key point of intersection between cellular metabolism and epigenetic gene regulation, and growing evidence supports its central role in several pathologies. The use of molecules that target NNMT represents a current pharmaceutical challenge for the treatment of several metabolic-related disease as well as in cancer.

Nicotinamide N-methyltransferase (NNMT), a key cytoplasmic protein in the human body, is accountable to catalyze the nicotinamide (NCA) N1-methylation through S-adenosyl-L-methionine (SAM) as a methyl donor, which has been linked to many diseases. Although extensive studies have concerned about the biological aspect, the detailed mechanism study of the enzyme function, especially in the part of protein dynamics is lacking. Here, wild-type nicotinamide N-methyltransferase together with the mutation at position 20 with Y20F, Y20G, and free tryptophan were carried out to explore the connection between protein dynamics and catalysis using time-resolved fluorescence lifetimes. The results show that wild-type nicotinamide N-methyltransferase prefers to adapt a less flexible protein conformation to achieve enzyme catalysis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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Background: The current study evaluated the level of serum N1-methylnicotinamide (me-NAM) in cervical cancer patients and further explored whether serum me-NAM was related to the prognosis of cervical cancer. Methods: Fifty-eight cases of cervical intraepithelial neoplasia patients, 78 cases of cervical cancer patients, and 52 healthy women were included in the present study. Serum me-NAM concentrations were determined by liquid chromatography with tandem mass spectrometry. Receiver operating characteristic (ROC) curve was used to assess me-NAM as a biomarker and Kaplan-Meier analysis was carried out to evaluate the survival rate. Results: Our data showed that the level of serum me-NAM in cervical cancer patients was significantly higher than that in the cervical intraepithelial neoplasia group and the healthy control group. Furthermore, the level of me-NAM in cervical cancer tissues of stage I, II, III, and IV was higher than that of those without lymph node metastasis. The area under the receiver operating characteristic curve (ROC) for me-NAM was higher than that of squamous cell carcinoma antigen (SCC Ag) and carbohydrate antigen 125 (CA125) when comparing cervical cancer from CIN or healthy control. The combination of me-NAM and SCC Ag or CA125 could improve the diagnostic efficiency better than SCC Ag or CA125 alone. Compared with me-NAM low expression group, the survival rate and time of me-NAM high expression group were lower and shorter. Conclusions: Altogether, elevated serum me-NAM levels contribute to the progression of cervical cancer and may be used as a marker for the prognosis of patients with cervical cancer. [ABSTRACT FROM AUTHOR]

Immune response to SARS-CoV-2 and ensuing inflammation pose a huge challenge to the host’s nicotinamide adenine dinucleotide (NAD+) metabolism. Humans depend on vitamin B3 for biosynthesis of NAD+, indispensable for many metabolic and NAD+-consuming signaling reactions. The balance between its utilization and resynthesis is vitally important. Many extra-pulmonary symptoms of COVID-19 strikingly resemble those of pellagra, vitamin B3 deficiency (e.g., diarrhoea, dermatitis, oral cavity and tongue manifestations, loss of smell and taste, mental confusion). In most developed countries, pellagra is successfully eradicated by vitamin B3 fortification programs. Thus, conceivably, it has not been suspected as a cause of COVID-19 symptoms. Here, the deregulation of the NAD+ metabolism in response to the SARS-CoV-2 infection is reviewed, with special emphasis on the differences in the NAD+ biosynthetic pathway’s efficiency in conditions predisposing for the development of serious COVID-19. SARS-CoV-2 infection-induced NAD+ depletion and the elevated levels of its metabolites contribute to the development of a systemic disease. Acute liberation of nicotinamide (NAM) in antiviral NAD+-consuming reactions potentiates “NAM drain”, cooperatively mediated by nicotinamide N-methyltransferase and aldehyde oxidase. “NAM drain” compromises the NAD+ salvage pathway’s fail-safe function. The robustness of the host’s NAD+ salvage pathway, prior to the SARS-CoV-2 infection, is an important determinant of COVID-19 severity and persistence of certain symptoms upon resolution of infection.

Ovarian cancer is the fifth leading cause of cancer-related mortality in women. Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme and there is growing evidence to suggest that it plays an important role in cancer progression. This is the first study to examine the expression of NNMT in serous ovarian cystadenomas, serous borderline tumours, low grade serous carcinomas (LGSC) and high grade serous carcinomas (HGSC) and investigate the potential independent association of NNMT expression with survival. Tissue samples were analysed immunohistochemically for NNMT expression. The stromal NNMT score was significantly higher in HGSC compared to serous cystadenomas and serous borderline tumours (p < .001, p < .043, respectively). The mean stromal NNMT score of patients with HGSC was significantly higher than patients with LGSC (p = .043). Patients with low expression of NNMT had a significantly higher mean recurrence-free survival than patients with high expression (p = .036). NNMT may support tumour progression in ovarian cancer by promoting desmoplastic stromal tumour reaction. NNMT overexpression may be associated with poor prognosis and can be a therapeutic target in ovarian cancer.IMPACT STATEMENTWhat is already known on this subject? Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme that is overexpressed in many malignancies. Its overexpression was shown to lead to histone hypomethylation, which in turn can decrease and increase the expression of tumour suppressor proteins and onco-proteins, respectively. NNMT was also shown to play a role in epithelial-to-mesenchymal transition, which is critical in tumour progression and the stromal tumour reaction. The stromal tumour reaction was recently targeted with promising therapeutic results in ovarian cancer.What do the results of this study add? The expression of NNMT in various ovarian neoplasms including serous cystadenomas, borderline tumours and serous carcinomas has not been studied and independently associated with poor survival, previously. This study suggests that NNMT is progressively overexpressed in the stroma of ovarian neoplasms from benign cysts to HGSCs. NNMT overexpression appears to be independently associated with poor survival in ovarian cancer.What are the implications of these findings for clinical practice and/or further research? The implications of these findings are that NNMT may play an important role in the stromal tumour reaction, and therefore its overexpression may contribute to poor survival. NNMT overexpression may be an important target of ovarian cancer therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nicotinamide N‐methyltransferase (NNMT) catalyzes the methyl transfer from universal methyl donor S‐adenosyl methionine (SAM) to nicotinamide (NA) to form the N‐methylnicotinamide. This important process is related to the level of nicotinamide adenine dinucleotide (NAD+) in vivo; thus, NNMT is regarded as an important drug target linked with various diseases. Although NNMT has been extensively studied in the area of biology and medicine, the detailed mechanism of NNMT is still not clear, especially in the aspect of the role of solvents (water) in the enzyme catalysis. Here, we have examined the effects of three common organic solvents (dimethyl sulfoxide [DMSO], methanol, and acetonitrile) and deuterated water to explore the enzymatic methyl transfer activity by NNMT (wild type [WT]) and its mutants. Competitive inhibition was detected for DMSO and acetonitrile as the solvent, and a subtle inhibitory effect was observed with methanol. Molecular docking suggested DMSO and acetonitrile compete with both cofactor and substrate. However, methanol is mainly bound to compete for the substrate. Furthermore, the activity increased when deuterated water was substituted for water with an inverse solvent isotope effect D2Okcat/Km = 0.49 ± 0.17 for WT and D2Okcat/Km = 0.32 ± 0.08 for Y20F. These effects in this enzymatic methyl transfer system without any metal ion or acid/base catalysis were due to the stabilization of protein provided by deuterated water. [ABSTRACT FROM AUTHOR]

The cancer stem cell theory states that a subset of tumor cells, termed cancer stem cells (CSCs), has the ability to self‐renew and differentiate within the tumors. According to this theory, CSCs would be mainly responsible for tumor initiation, progression, resistance to therapy, recurrence, and metastasis. In this study, a culture system was setup to enrich CSCs from bladder cancer (T24), lung cancer (A549), colorectal cancer (CaCo‐2), and osteosarcoma (MG63) cell lines, through sphere formation. Magnetic‐activated cell sorting was also used to further increase CSC enrichment. Subsequently, molecular characterization of CSC‐enriched cell populations and parental cells was carried out, by exploring the expression levels of stem markers and the enzyme nicotinamide N‐methyltransferase (NNMT). Results obtained showed a significant upregulation of stem cell markers in CSC‐enriched populations, obtained upon sphere formation, compared with parental counterparts. Moreover, NNMT expression levels were markedly increased in samples enriched with CSCs with respect to control cells. Considering the fundamental role played by CSCs in carcinogenesis, reported data strengthen the hypothesis that sustains a pivotal role of NNMT in cancer growth and metastasis. In addition, these findings could represent an important achievement for the development of new and effective anticancer therapies, based on CSC‐associated targets. [ABSTRACT FROM AUTHOR]

Background: Nicotinamide N-methyltransferase (NNMT) is highly expressed in several cancers and can regulate cell epigenetic status and various cell metabolism pathways, such as ATP synthesis and cellular stress response. We reported in our previous papers that NNMT overexpression inhibits the apoptosis and enhances the chemotherapy resistance of breast cancer cells. This study aims to investigate the effect of NNMT on autophagy induced by oxidative stress in breast cancer cells, which might provide a novel therapeutic strategy for breast cancer treatment. Methods: NNMT and LC3B II protein levels in the two cell models (SK-BR-3 and MDA-MB-231) with NNMT overexpression or knockdown were detected by Western blotting and correlated with each other. Changes in cellular viability, intracellular reactive oxygen species (ROS) and ATP levels were assessed after H2O2 treatment. Then, autophagosomes were imaged by transmission electron microscopy, and LC3 puncta were examined by confocal microscopy and flow cytometry. The LC3B II level and AMPK-ULK1 pathway activity were both detected by Western blotting to determine the role of NNMT in the H2O2-induced autophagy. Results: NNMT expression was negatively correlated with LC3B II expression in both cell models (SK-BR-3 and MDA-MB-231). Then, NNMT overexpression attenuated the autophagy induced by H2O2 in SK-BR-3 cells, whereas knockdown promoted autophagy induced by H2O2 in MDA-MB-231 cells. Furthermore, mechanistic studies showed that NNMT suppressed the ROS increase, ATP decrease and AMPK-ULK1 pathway activation, resulting in the inhibition of H2O2-induced autophagy in breast cancer cells. Conclusions: We conclude that NNMT inhibits the autophagy induced by oxidative stress through the ROS-mediated AMPK-ULK1 pathway in breast cancer cells and may protect breast cancer cells against oxidative stress through autophagy suppression. [ABSTRACT FROM AUTHOR]

[Display omitted] • A series of bisubstrate NNMT inhibitors are designed and synthesized. • Modifications at the N7 site of deazaadenine with various substitutions are well tolerated and lead to novel potent NNMT inhibitors. • Among the analogs, compound 3–12 demonstrated potent NNMT inhibitory activity as well as good selectivity over a panel of methyltransferases. Nicotinamide N -methyltransferase (NNMT) catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to nicotinamide (NAM) and other pyridine-related compounds and is involved in various metabolic processes in the human body. In addition, abnormal expression of NNMT occurs under various pathological conditions such as cancer, diabetes, metabolic disorders, and neurodegenerative diseases, making it a promising drug target worthy of in-depth research. Small-molecule NNMT inhibitors with high potency and selectivity are necessary chemical tools to test biological hypotheses and potential therapies. In this study, we developed a series of highly active NNMT inhibitors by modifying N7 position of adenine. Among them, compound 3 – 12 (IC 50 = 47.9 ± 0.6 nM) exhibited potent inhibitory activity and also had an excellent selectivity profile over a panel of human methyltransferases. We showed that the N7 position of adenine in the NNMT bisubstrate inhibitor was a modifiable site, thus offering insights into the development of NNMT inhibitors. [ABSTRACT FROM AUTHOR]

The head and neck tumors (HNT) are a heterogeneous group of diseases ranging from benign to malignant lesions, with distinctive molecular and clinical behaviors. Several studies have highlighted the presence of an altered metabolic phenotype in HNT, such as the upregulation of nicotinamide N-methyltransferase (NNMT). However, its biological effects have not been completely disclosed and the role of NNMT in cancer cell metabolism remains unclear. Therefore, this comprehensive review aims to evaluate the available literature regarding the biological, diagnostic, and prognostic role of NNMT in HNT. NNMT was shown to be significantly overexpressed in all of the evaluated HNT types. Moreover, its upregulation has been correlated with cancer cell migration and adverse clinical outcomes, such as high-pathological stage, lymph node metastasis, and locoregional recurrences. However, in oral squamous cell carcinoma (OSCC) these associations are still debated, and several studies have failed to demonstrate the prognostic significance of NNMT. The shRNA-mediated gene silencing efficiently suppressed the NNMT gene expression and exhibited a clear inhibitory effect on cell proliferation, promoting the expression of apoptosis-related proteins and modulating the cell cycle. NNMT could represent a new molecular biomarker and a new target of molecular-based therapy, although further studies on larger patient cohorts are needed to explore its biological role in HNT.

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation reaction of nicotinamide, using S-adenosyl-L-methionine as the methyl donor. Enzyme overexpression has been described in many non-neoplastic diseases, as well as in a wide range of solid malignancies. This review aims to report and discuss evidence available in scientific literature, dealing with NNMT expression and the potential involvement in main urologic neoplasms, namely, renal, bladder and prostate cancers. Data illustrated in the cited studies clearly demonstrated NNMT upregulation (pathological vs. normal tissue) in association with these aforementioned tumors. In addition to this, enzyme levels were also found to correlate with key prognostic parameters and patient survival. Interestingly, NNMT overexpression also emerged in peripheral body fluids, such as blood and urine, thus leading to candidate the enzyme as promising biomarker for the early and non-invasive detection of these cancers. Examined results undoubtedly showed NNMT as having the capacity to promote cell proliferation, migration and invasiveness, as well as its potential participation in fundamental events highlighting cancer progression, metastasis and resistance to chemo- and radiotherapy. In the light of this evidence, it is reasonable to attribute to NNMT a promising role as a potential biomarker for the diagnosis and prognosis of urologic neoplasms, as well as a molecular target for effective anti-cancer treatment.

Nicotinamide N-methyltransferase (NNMT) plays multiple roles in improving the aggressiveness of colorectal cancer (CRC) and enhancing resistance to 5-Fluorouracil (5-FU), making it an attractive therapeutic target. Curcumin (Cur) is a promising natural compound, exhibiting multiple antitumor effects and potentiating the effect of 5-FU. The aim of the present study is to explore the effect of Cur on attenuating NNMT-induced resistance to 5-FU in CRC. A panel of CRC cell lines with different NNMT expressions are used to characterize the effect of Cur. Herein, it is observed that Cur can depress the expression of NNMT and p-STAT3 in CRC cells. Furthermore, Cur can induce inhibition of cell proliferation, G2/M phase cell cycle arrest, and reactive oxygen species (ROS) generation, especially in high-NNMT-expression CRC cell lines. Cur can also re-sensitize high-NNMT-expression CRC cells to 5-FU both in vitro and in vivo. In summary, it is proposed that Cur can reverse NNMT-induced cell proliferation and 5-FU resistance through ROS generation and cell cycle arrest. Given that Cur has long been used, we suppose that Cur is a promising anticancer drug candidate with minimal side effects for human CRC therapy and can attenuate NNMT-induced resistance to 5-FU.

The activity of nicotinamide N-methyltransferase (NNMT) is tightly linked to the maintenance of the nicotinamide adenine dinucleotide (NAD+) level. This enzyme catalyzes methylation of nicotinamide (NAM) into methyl nicotinamide (MNAM), which is either excreted or further metabolized to N1-methyl-2-pyridone-5-carboxamide (2-PY) and H2O2. Enzymatic activity of NNMT is important for the prevention of NAM-mediated inhibition of NAD+-consuming enzymes poly–adenosine -diphosphate (ADP), ribose polymerases (PARPs), and sirtuins (SIRTs). Inappropriately high expression and activity of NNMT, commonly present in various types of cancer, has the potential to disrupt NAD+ homeostasis and cellular methylation potential. Largely overlooked, in the context of cancer, is the inhibitory effect of 2-PY on PARP-1 activity, which abrogates NNMT’s positive effect on cellular NAD+ flux by stalling liberation of NAM and reducing NAD+ synthesis in the salvage pathway. This review describes, and discusses, the mechanisms by which NNMT promotes NAD+ depletion and epigenetic reprogramming, leading to the development of metabolic plasticity, evasion of a major tumor suppressive process of cellular senescence, and acquisition of stem cell properties. All these phenomena are related to therapy resistance and worse clinical outcomes.

Background/aim: Nicotinamide N-methyltransferase (NNMT) is an enzyme that is overexpressed in malignancies. NNMT expression has not been previously studied in endometrial cancer (EC). Increased phospho-Akt (pAkt) levels in response to NNMT overexpression have been reported in in vitro studies of different cancer types. We assayed NNMT expression in primary and metastatic high-grade EC and investigated the relationship of NNMT with p53, pAkt, and survival. Materials and methods: NNMT, pAkt, and p53 expressions were assayed in 100 tissue samples of benign endometria, primary EC, and metastatic EC by immunohistochemistry. Results: The NNMT immunoreactivity score was significantly higher in primary high-grade EC than benign endometrial tissue (P = 0.001). NNMT expression in metastatic tissue was significantly higher than in primary cancer (P < 0.001). Metastatic stromal NNMT expression was significantly higher than that of the adjacent tumor and stroma adjacent to the primary tumor. p53 expression in the primary tumor showed a significant positive correlation with omental NNMT and pAkt expression. NNMT expression was also correlated with pAkt expression in metastatic tissue. NNMT overexpression in metastatic tissue was associated with decreased survival (P = 0.039). Conclusion: This study suggests that NNMT may promote cancer progression and that NNMT overexpression is associated with aberrant p53 expression, pAkt, and poor survival. NNMT's role in cancer progression could make it a target of EC therapy. [ABSTRACT FROM AUTHOR]