Tumor stromal nicotinamide N-methyltransferase overexpression as a prognostic biomarker for poor clinical outcome in early-stage colorectal cancer.

In a quest for prognostic biomarkers in early-stage colorectal cancer, we investigated NNMT (nicotinamide N-methyltransferase) in large cohorts of patients. Immunohistochemical examination of 679 patients illustrates that NNMT protein is predominantly expressed in the cancer stroma at varying levels, and about 20% of cancer tissues overexpress NNMT when compared to levels observed in normal colorectal mucosa. Clinical correlation analyses of 572 patients with early-stage cancers reveal that NNMT protein overexpression is significantly associated with shorter overall and disease-free survival, but no such correlation is found in late-stage colorectal cancer. Analyses of TCGA and CPTAC colorectal cancer cohorts show that NNMT mRNA expression is positively correlated with protein levels, is significantly higher in CIMP-high or MSI subtypes than in CIMP-low or MSS subtypes, and is positively correlated with its paralog INMT but not with its interaction partners such as PNMT, ADK, APP, ATF6, BMF, BRD4, CDC37, or CRYZ. In early-stage cancers, NNMT expression is higher in BRAF-mutated than in BRAF wild type tumors but is not affected by KRAS or PIK3CA mutation status. As a cancer stromal protein with important roles in metabolism and cancer epigenetics, NNMT is emerging as a promising biomarker for risk stratification of early-stage cancers.

Nicotinamide N-methyltransferase gene silencing enhances chemosensitivity of melanoma cell lines.

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.

ER stress-induced upregulation of NNMT contributes to alcohol-related fatty liver development.

BACKGROUND & AIMS: N-nicotinamide methyltransferase (NNMT) is emerging as an important enzyme in the regulation of metabolism. NNMT is highly expressed in the liver. However, the exact regulatory mechanism(s) underlying NNMT expression remains unclear and its potential involvement in alcohol-related liver disease (ALD) is completely unknown. METHODS: Both traditional Lieber-De Carli and the NIAAA mouse models of ALD were employed. A small-scale chemical screening assay and a chromatin immunoprecipitation assay were performed. NNMT inhibition was achieved via both genetic (adenoviral short hairpin RNA delivery) and pharmacological approaches. RESULTS: Chronic alcohol consumption induces endoplasmic reticulum (ER) stress and upregulates NNMT expression in the liver. ER stress inducers upregulated NNMT expression in both AML12 hepatocytes and mice. PERK-ATF4 pathway activation is the main contributor to ER stress-mediated NNMT upregulation in the liver. Alcohol consumption fails to upregulate NNMT in liver-specific Atf4 knockout mice. Both adenoviral NNMT knockdown and NNMT inhibitor administration prevented fatty liver development in response to chronic alcohol feeding; this was also associated with the downregulation of an array of genes involved in de novo lipogenesis, including Srebf1, Acaca, Acacb and Fasn. Further investigations revealed that activation of the lipogenic pathway by NNMT was independent of its NAD+-enhancing action; however, increased cellular NAD+, resulting from NNMT inhibition, was associated with marked liver AMPK activation. CONCLUSIONS: ER stress, specifically PERK-ATF4 pathway activation, is mechanistically involved in hepatic NNMT upregulation in response to chronic alcohol exposure. Overexpression of NNMT in the liver plays an important role in the pathogenesis of ALD. LAY SUMMARY: In this study, we show that nicotinamide methyltransferase (NNMT) - the enzyme that catalyzes nicotinamide degradation - is a pathological regulator of alcohol-related fatty liver development. NNMT inhibition protects against alcohol-induced fatty liver development and is associated with suppressed de novo lipogenic activity and enhanced AMPK activation. Thus, our data suggest that NNMT may be a potential therapeutic target for the treatment of alcohol-related liver disease.

Nicotinamide N-methyltransferase expression in SH-SY5Y human neuroblastoma cells decreases oxidative stress.

Nicotinamide N-methyltransferase (NNMT) plays a central role in cellular metabolism, regulating pathways including epigenetic regulation, cell signalling, and energy production. Our previous studies have shown that the expression of NNMT in the human neuroblastoma cell line SH-SY5Y increased complex I activity and subsequent ATP synthesis. This increase in ATP synthesis was lower than the increase in complex I activity, suggesting uncoupling of the mitochondrial respiratory chain. We, therefore, hypothesised that pathways that reduce oxidative stress are also increased in NNMT-expressing SH-Y5Y cells. The expression of uncoupling protein-2 messenger RNA and protein were significantly increased in NNMT-expressing cells (57% ± 5.2% and 20.1% ± 1.5%, respectively; P = .001 for both). Total GSH (22 ± 0.3 vs 35.6 ± 1.1 nmol/mg protein), free GSH (21.9 ± 0.2 vs 33.5 ± 1 nmol/mg protein), and GSSG (0.6 ± 0.02 vs 1 ± 0.05 nmol/mg protein; P = .001 for all) concentrations were significantly increased in NNMT-expressing cells, whereas the GSH:GSSG ratio was decreased (39.4 ± 1.8 vs 32.3 ± 2.5; P = .02). Finally, reactive oxygen species (ROS) content was decreased in NNMT-expressing cells (0.3 ± 0.08 vs 0.12 ± 0.03; P = .039), as was the concentration of 8-isoprostane F2α (200 ± 11.5 vs 45 ± 2.6 pg/mg protein; P = .0012). Taken together, these results suggest that NNMT expression reduced ROS generation and subsequent lipid peroxidation by uncoupling the mitochondrial membrane potential and increasing GSH buffering capacity, most likely to compensate for increased complex I activity and ATP production.

Expression and Clinical Significance of Nicotinamide N-Methyltransferase in Cervical Squamous Cell Carcinoma.

Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme, overexpressed in various human malignancies. It is associated with cancer progression and resistance to treatment. The role of NNMT in cervical cancer has not been studied thus far. We aimed to evaluate expression of NNMT in cervical squamous cell carcinoma (SCC) and investigate its clinical significance. NNMT expression was assayed by use of immunohistochemistry in 61 cases of SCC, 11 cases of high-grade squamous intraepithelial lesion, 17 cases of low-grade squamous intraepithelial lesion, and 51 benign cervical tissues. NNMT immunoreactivity was scored based on staining intensity and percentage of positively stained cells. The expression of NNMT was significantly higher in SCC than in benign tissue, low-grade squamous intraepithelial lesion, and high-grade squamous intraepithelial lesion (P<0.001). NNMT expression in benign tissue was significantly lower than in low-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion. When stratified according to stage, NNMT expression was significantly higher in patients with stage III and IV than those in stage I and II disease (P=0.009). For all stages, patients with metastatic pelvic or para-aortic lymph nodes had significantly higher NNMT expression than patients without nodal involvement (P=0.001). Although preliminary, this is the first study to detect overexpression of NNMT in SCC and increased expression associated with advanced stage and metastatic lymph nodes. NNMT should be investigated further in cervical cancer as a potential therapeutic target and a prognostic indicator.

Nicotinamide N-methyltransferase expression and its association with phospho-Akt, p53 expression, and survival in high-grade endometrial cancer

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.

Elevated N-methyltransferase expression induced by hepatic stellate cells contributes to the metastasis of hepatocellular carcinoma via regulation of the CD44v3 isoform.

The cross-talk between hepatic stellate cells (HSCs) and hepatic carcinoma cells contributes to hepatocellular carcinoma (HCC) progression, but the underlying mechanism is largely unknown. We report here that activated HSCs induce upregulation of nicotinamide N-methyltransferase (NNMT), which is known to regulate multiple metabolic pathways in hepatoma cells of the liver. High levels of NNMT in HCC tissues were positively correlated with vascular invasion, increased serum HBV-DNA levels, and distant metastasis. In addition, functional assays showed that NNMT promoted HCC cell invasion and metastasis by altering the histone H3 methylation on 27 methylation pattern and transcriptionally activating cluster of differentiation 44 (CD44). NNMT-mediated N6-methyladenosine modification of CD44 mRNA resulted in the formation of a CD44v3 splice variant, while its product 1-methyl-nicotinamide stabilized CD44 protein by preventing ubiquitin-mediated degradation. Finally, NNMT was also shown to be a target of statins that inhibited metastasis of hepatoma cells. Taken together, our study shows for the first time that the NNMT/CD44v3 axis regulates HCC metastasis and presents NNMT as a promising prognostic biomarker and therapeutic target for HCC.

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.

Role of Nicotinamide N-Methyltransferase in Dorsal Striatum in Cocaine Place Preference.

Nicotinamide N-methyltransferase (NNMT) transfers the methyl from S-adenosyl-L-methionine (SAM) to nicotinamide (NA) to produce S-adenosyl-L-homocysteine (SAH) and 1-methylnicotinamide (MeN). NNMT has been implicated in a variety of diseases; however, the role of NNMT in drug addiction is largely unknown. Here, we found that the expression of Nnmt was significantly upregulated in the dorsal striatum (DS) of cocaine-conditioned mice. Cocaine significantly decreased SAM/SAH ratio levels in the DS, which was accompanied with the decreased activities of Rac1 and RhoA. Lentivirus-mediated knockdown of Nnmt in the dorsomedial striatum (DMS) attenuated cocaine conditioned place preference (CPP) reward, but increased striatal SAM/SAH ratio levels as well as Rac1 and RhoA activities. In addition, pharmacological inhibition of NNMT through intra-DMS infusion of MeN attenuated cocaine CPP and the activities of Rac1 and RhoA, but increased SAM/SAH ratio. These results suggest that NNMT-dependent transmethylation is involved in the activation of Rac1 and RhoA, which utilize SAM as a methyl donor cofactor. Co-immunoprecipitation assay using a RhoGDIα antibody indirectly captured Rac1 or RhoA that were bound to RhoGDIα. The results showed that cocaine increased the association of RhoGDIα with Rac1 or RhoA, whereas such effect was inhibited by Nnmt knockdown. Collectively, our findings show that NNMT regulates cocaine CPP through SAM-mediated modification of Rac1 and RhoA.

Nicotinamide N-methyltransferase enhances the capacity of tumorigenesis associated with the promotion of cell cycle progression in human colorectal cancer cells.

Nicotinamide N-methyltransferase (NNMT), an enzyme involved in the biotransformation and detoxification of many drugs and xenobiotic compounds, has been found to be overexpressed in several malignancies, including colorectal cancer. However, the biological function of NNMT and the related mechanisms in colorectal cancer have not been elucidated. In the present study, we investigated the effects of NNMT on tumorigenesis by overexpressing NNMT in the human colorectal cancer cells line SW480 which lacks constitutive NNMT expression, and downregulating NNMT expression in HT-29 cells, which exhibit high endogenous expression of NNMT. We found that NNMT significantly accelerates cell proliferation, enhances colony formation in vitro and tumorigenicity in mice; it also inhibits apoptosis, promotes cell cycle progression, increases ATP and 1-methylnicotinamide level and decreases ROS level. We also showed that 1-methylnicotinamide accelerates cell growth, inhibits apoptosis, promotes cell cycle progression, attenuates ROS production and increases ATP level. Our results indicate that NNMT enhances the capacity of tumorigenesis associated with the inhibition of cell apoptosis and the promotion of cell cycle progression in human colorectal cancer cells and the 1-methylnicotinamide increased by NNMT mediates the cellular effects of NNMT in cells. NNMT may play a vital role in energy balance and ROS induction.

Neuroprotective effects of nicotinamide N-methyltransferase and its metabolite 1-methylnicotinamide.

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) catalyses the N-methylation of nicotinamide to 1-methylnicotinamide (MeN). We have previously shown that the ectopic expression of NNMT in SH-SY5Y human neuroblastoma cells increased adenosine triphosphate synthesis and complex I activity, effects of which were replicated by the addition of MeN. In this study, we investigated whether NNMT expression in SH-SY5Y conferred protection against mitotoxicity induced by rotenone, potassium cyanide (KCN), 2,4-dinitrophenol, and 6-hydroxydopamine, and whether any effects observed were mediated via increased MeN production. NNMT expression abolished the toxic effects of KCN, 2,4-dinitrophenol, and 6-hydroxydopamine, and reduced that of rotenone. In contrast, although MeN significantly reduced the toxicity of rotenone, it had no effect upon the toxicity of KCN, 2,4-dinitrophenol, and 6-hydroxydopamine. These data show that NNMT is cytoprotective against toxins that inhibit various aspects of mitochondrial function, and that these are not mediated solely via increased MeN production, but in combination with other unidentified mechanisms.

Nicotinamide N-Methyltransferase upregulation correlates with tumour differentiation in oral squamous cell carcinoma.

We investigated expression levels of Nicotinamide N-Methyltransferase (NNMT), an enzyme involved in the biotransformation of many drugs and xenobiotic compounds, in oral squamous cell carcinoma (OSCC). Measurements were performed by immunohistochemistry and the relationship between tumour characteristics and NNMT levels in OSCC was studied to evaluate the effectiveness of NNMT as a prognostic marker in squamous cell carcinoma of the oral cavity. In conclusion, the present study suggests that NNMT may have potential as a biomarker and as a therapeutic target for OSCC.

Expression of nicotinamide N-methyltransferase in hepatocellular carcinoma is associated with poor prognosis.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common tumor in the adult liver, with high relapse and mortality rates despite diverse treatment modalities. In this study, nicotinamide N-methyltransferase (NNMT), a key enzyme in drug metabolism, was investigated as a potential prognostic factor. METHODS: Frozen tumors and non-cancerous surrounding tissues from 120 patients with primary HCC were studied. Expressions of NNMT and internal control genes were measured by real-time reverse-transcription PCR (RT-PCR). The relationship of NNMT mRNA level with clinicopathologic parameters and clinical outcome was evaluated. RESULTS: NNMT mRNA level is markedly reduced in HCCs compared to non-cancerous surrounding tissues (P < 0.0001), and NNMT expression in tumors was significantly correlated with tumor stage (P = 0.010). Moreover, stratification of patients based on tumor NNMT mRNA levels revealed that the patients who expressed higher NNMT mRNA levels tended to have a shorter overall survival (OS) time (P = 0.053) and a significantly shorter disease-free survival (DFS) time (P = 0.016). Both NNMT expression (P = 0.0096) and tumor stage (P = 0.0017) were found to be significant prognostic factors for DFS in a multivariate analysis. CONCLUSION: The results of this study indicated that NNMT gene expression is associated with tumor stage and DFS time in HCC cases. Because of the broad substrate specificity of NNMT, which could alter the efficacy and adverse effects of chemotherapy, NNMT merits further investigation regarding its role as a prognostic factor with a larger cohort of HCC patients.

Serum levels of nicotinamide N-methyltransferase in patients with lung cancer.

PURPOSE: The aim of this study was to determine if nicotinamide N-methyltransferase (NNMT) is useful as a biomarker of lung cancer (stages I-III). METHODS: We established an ELISA system for NNMT. We determined the levels of NNMT and carcinoembryonic antigen (CEA) in sera of 113 non-small cell lung cancer (NSCLC) patients undergoing surgery and sera of 50 non-neoplastic lung disease patients with chronic obstructive pulmonary disease (COPD) and of 24 healthy donors. RESULTS: The serum levels of NNMT were significantly higher in lung cancer patients than in COPD patients and healthy donors. The relationship between the specificity and sensitivity of NNMT and CEA measurements for the detection of lung cancer was analyzed by means of receiver-operating characteristic curves. The corresponding areas under the curves were 0.703 for NNMT and 0.621 for CEA, indicating slightly better sensitivity of NNMT. With 90% specificity, the sensitivities of NNMT and CEA as lung cancer markers were 25 and 24%, respectively. There was no significant correlation between NNMT and CEA. Therefore, the sensitivity of NSCLC detection at 90% specificity increased from 25 to 32% when NNMT was used in combination with CEA. CONCLUSION: The NNMT serum level may have significance in the early detection of NSCLC patients.

Stat3 up-regulates expression of nicotinamide N-methyltransferase in human cancer cells.

PURPOSE: To discover new molecular targets for cancer therapy and diagnosis, we surveyed signal transducers and activators of transcription 3 (Stat3)-regulated genes, because constitutive activation of Stat3 is associated with a wide variety of human malignancies. METHODS: We investigated the Stat3-regulated genes in 293 cells with cDNA microarray analysis and found that Nicotinamide N-methyltransferase (NNMT) was induced on stimulation of the cells with leukemia inhibitory factor. We examined the expression of NNMT in several types of cancer cells by real-time quantitative RT-PCR. To examine the role of Stat3, Hep-G2 hepatocellular carcinoma cells were transfected with NNMT promoter-luciferase reporter construct together with conditionally active Stat3 (Stat3ER) or dominant-negative Stat3 expression vector and NNMT promoter activity was determined. The expression of NNMT and activated Stat3 in 88 colon cancer tissues and 17 normal colon tissues was examined with immunohistochemical analysis. RESULTS: In Hep-G2 cells and SW480 colon cancer cells, NNMT expression increased on stimulation of the cells with interleukin 6. NNMT promoter activity in Hep-G2 cells was dependent on the activation of Stat3. MDA-MB-468 breast cancer cells and HT29 colon cancer cells expressed constitutively a high level of NNMT. Treatment of these cells with Stat3 siRNA or curcumin, which inhibited Stat3 phosphorylation, resulted in reduction of the NNMT level. We found a correlation between the expression of NNMT and activated Stat3 (P<0.001) in the colon cancer tissues. CONCLUSION: NNMT is a novel Stat3-regulated gene. Its expression is enhanced with the activation of Stat3 in colon cancer tissues. NNMT may be a potential candidate for a tumor marker of various kinds of cancers.

Identification of nicotinamide N-methyltransferase as a novel tumor marker for renal clear cell carcinoma.

PURPOSE: To explore the involvement of enzymes of drug metabolism in renal cell carcinoma we analyzed the gene expression profiles of tumor and nontumor tissues from the same patient by DNA macroarray. The enzyme nicotinamide N-methyltransferase was selected for further evaluation. MATERIALS AND METHODS: Nicotinamide N-methyltransferase mRNA expression was investigated in paired tissue samples from cancerous and noncancerous parts of the kidneys of 30 patients with clear cell renal cell carcinoma who underwent tumor nephrectomy. Measurements were performed by semiquantitative reverse transcriptase-polymerase chain reaction and quantitative real-time polymerase chain reaction. Paired tissue samples were also obtained from 1 patient with chromophobe renal cell carcinoma and from another with oncocytoma to compare the specificity of changes in nicotinamide N-methyltransferase expression among tumors that are related to different renal epithelial cell types. Western blot analysis and catalytic activity assay were also performed to study nicotinamide N-methyltransferase expression. Expression correlated with tumor characteristics. RESULTS: A marked increased expression in tumor tissue was found for nicotinamide N-methyltransferase, which is an enzyme involved in the biotransformation of many drugs and xenobiotic compounds. Differential gene expression measurements in tumor vs normal tissue revealed up-regulation in all clear cell renal cell carcinomas at between 3 and 294-fold (mean 41). In contrast, in chromophobe renal cell carcinoma and oncocytoma nicotinamide N-methyltransferase expression did not increase. In addition, nicotinamide N-methyltransferase expression significantly correlated inversely with tumor size. CONCLUSIONS: Our results indicate that a marked nicotinamide N-methyltransferase increase is a peculiar feature of clear cell renal cell carcinoma. Additional studies may establish the role of nicotinamide N-methyltransferase in tumor formation and progression.