Nicotinamide N-Methyltransferase (NNMT) and Liver Cancer: From Metabolic Networks to Therapeutic Targets

Primary liver cancer (PLC) ranks as the sixth most prevalent type of cancer globally and the third major contributor to cancer-related mortality [ 1 ]. PLC primarily consists of three types: intrahepatic cholangiocarcinoma (ICC), hepatocellular carcinoma (HCC), and combined hepatocellular-cholangiocarcinoma (cHCC-CCA). HCC constitutes approximately 90% of primary liver cancer cases [ 2 ], exhibiting both high incidence and mortality rates [ 3 ]. Growing evidence suggests that early stages of HCC development and its precursor lesions share common features regulated by genetic and epigenetic mechanisms [ 4 , 5 ]. Additionally, alterations in various methylation processes have been observed in liver diseases, including an increase in nicotinamide methylation in cirrhosis patients [ 6 , 7 , 8 , 9 ]. Therefore, gaining a comprehensive insight into the etiology of liver cancer and pinpointing viable therapeutic targets are crucial steps in improving the survival rates and overall well-being of patients suffering from this disease. Emerging evidence highlights the dual role of Nicotinamide N-methyltransferase (NNMT) across solid tumors, with both conserved and tissue-specific mechanisms. In breast cancer, NNMT stabilizes SIRT1 to drive chemoresistance [ 10 ], while, in pancreatic cancer, it paradoxically suppresses tumorigenesis by altering oncometabolite profiles [ 11 ]. Notably, NNMT’s impact on NAD + depletion and methionine cycle dysregulation is a shared hallmark in cancers such as HCC and ovarian tumors [ 12 ]; yet, its functional outcomes diverge depending on cellular context. For instance, NNMT promotes immune evasion in HCC through H3K27me3-mediated CD44 upregulation, whereas, in melanoma, it primarily induces chemoresistance via epigenetic silencing [ 13 ]. These comparisons underscore the need to dissect NNMT’s role through both pan-cancer and tissue-specific lenses, particularly in liver cancer where viral hepatitis and unique methionine cycle dependencies amplify its pro-tumorigenic effects. Elevated NNMT levels have been observed in various liver cancers, and this upregulation is linked to cancer growth and progression [ 14 , 15 , 16 ]. NNMT, a key metabolic enzyme, regulates the levels of S-adenosyl-L-homocysteine (SAH), N 1 -methylnicotinamide (MNAM), S-adenosyl-L-methionine (SAM), and nicotinamide (NAM) in liver cancer cells. It plays a crucial role in NAD + -mediated signaling pathways and homocysteine (Hcy) metabolism by utilizing NAM, a precursor to NAD + , and producing SAH, which is a precursor to Hcy [ 17 , 18 ]. NNMT dysregulation is implicated in multiple pathological conditions. In breast cancer, a high NNMT expression correlates with poor survival and adverse chemotherapy responses [ 10 ]. In pancreatic cancer, it modulates the metabolome to suppress malignant behaviors and tumorigenesis [ 11 ]. NNMT inhibition improves metabolic parameters, including energy expenditure, fat accumulation, insulin sensitivity, glucose tolerance, and fasting glucose [ 19 ]. In neurodegenerative diseases, NNMT dysregulation drives pathogenesis via NAD + depletion and metabolic disruption [ 20 ]. Although the specific mechanisms by which NNMT influences liver cancer progression and its potential as a therapeutic target remain poorly understood, accumulating evidence suggests that NNMT plays significant roles in diagnosis, treatment, and prognosis. This review delves into the regulatory mechanisms governing NNMT expression in liver cancer and its association with malignant behaviors.

It is well-established that cancer cells modify their metabolic processes to support rapid cell division and survive in unfavorable conditions such as hypoxia and nutrient scarcity [ 41 ]. Cancer cells, in contrast to their normal counterparts, display elevated ratios of oxidized nicotinamide adenine dinucleotide phosphate (NADP + ) to NADPH, as well as NAD + to NADH, emphasizing the pivotal role of NAD + in the metabolic shift that defines cancer metabolism [ 42 ]. Tumor cells often adapt to stressful environments and achieve unlimited proliferation through metabolic reprogramming, a characteristic that holds potential for non-invasive cancer diagnosis [ 43 , 44 ]. There are significant differences in energy acquisition between tumor cells and normal cells. While normal cells predominantly rely on oxidative phosphorylation (OXPHOS) for energy production, cancer cells often prefer glycolysis as their primary energy source, even when ample oxygen is available, a metabolic adaptation referred to as the “Warburg effect” ( Figure 2 ) [ 45 ]. The Warburg effect [ 46 ] delineates the preferred metabolic route of cancer cells, favoring glycolysis over OXPHOS, even in the presence of abundant oxygen. Cancer cells predominantly utilize glucose for energy production and are reliant on NAD + to catalyze essential metabolic processes [ 25 ]. Additionally, several mechanisms promote the development of liver cancer, including genetic mutations, pathway activation, and somatic DNA alterations in liver cancer cells, such as mutations in the TP53 [ 47 ], TERT promoter [ 48 ], and genes involved in WNT signaling [ 49 ]. These changes affect critical cellular functions such as the control of cell division, differentiation, and apoptosis. Immune evasion also plays a central role in liver cancer progression. Metabolic reprogramming within the tumor microenvironment (TME) meets the energy demands of both cancer cells and the immune components of the tumor, thereby driving disease progression. Tumor cells promote rapid proliferation by upregulating glycolysis, gluconeogenesis, and β-oxidation, resulting in a glucose-deficient but lactate-rich environment that alters immune responses in the liver, enabling immune evasion by tumor cells [ 50 ]. Consequently, neoplastic cells markedly enhance their glucose intake to support their unchecked growth and proliferation [ 51 , 52 ]. For the maintenance of elevated glycolytic rates, it is crucial that we increase NAD + levels, considering its critical function in key metabolic processes. For instance, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the transformation of glyceraldehyde-3-phosphate into lactate. Although OXPHOS generates ATP more efficiently (producing 36 ATP molecules per mole of glucose), cancer cells tend to favor glycolysis due to their higher ATP demand, producing only two ATP molecules per mole of glucose [ 53 ]. The swift generation of energy grants hepatic cancer cells a competitive edge in environments where nutrients are scarce, enabling them to proliferate more rapidly than the surrounding stromal cells. Furthermore, the lactate generated through glycolysis leads to the acidification of the tumor microenvironment, a condition that fosters the invasiveness of cancer cells and dampens immune responses against the tumor. NAM, acting as a precursor for NAD + , potently inhibits sirtuin enzymes. NNMT regulates the synthesis of NAD + through the metabolic conversion of NAM. Empirical evidence indicates that NNMT has the ability to enhance the expression and activation of sirtuins [ 54 ]. Pioneering research by Estep et al. [ 55 ] initially revealed the link between NNMT and the sirtuin family, influencing both their expression and functionality. They observed increased levels of NNMT and Sirt1 expression following caloric restriction in mice. Further studies [ 56 ] have indicated that the activation of Sirt1 promotes gluconeogenesis, inhibits cholesterol synthesis, and suppresses lipogenesis, implying that NNMT could be pivotal in modulating these metabolic pathways through the stabilization of the Sirt1 protein [ 57 ]. B et al. [ 58 ] demonstrated that SIRT1 and SIRT3 are essential for enhancing mitochondrial performance under conditions of glucose scarcity. This enhancement is mediated through the activation of the AMPK-p53-PGC1α signaling axis in HCC cells. This highlights the metabolic role of SIRT1 in HCC pathogenesis and suggests its potential relevance for future liver cancer treatments. Conversely, the addition of NAD + precursors, including NA or NAM, may counteract these metabolic alterations, facilitating the re-establishment of cellular homeostasis. Additionally, Zhang et al. [ 59 ] investigated SIRT6 levels across a range of HCC cell lines, delving into its influence on cellular expansion and programmed cell death. By transfecting Huh-7 cells with plasmids for SIRT6 overexpression and small interfering RNA (siRNA) for silencing SIRT6, they found

The disruption of Hcy metabolism may increase susceptibility to diseases like HCC [ 87 ]. A substantial body of research has repeatedly demonstrated a significant association between increased Hcy levels and the occurrence of cancer [ 88 ], including liver cancer. First, cancer patients typically have elevated plasma Hcy levels. Second, genetic variations in enzymes that participate in Hcy detoxification, including transsulfuration and remethylation, are closely associated with various types of cancer. Third, folate, indispensable for cellular proliferation, is inversely associated with Hcy levels. In the fourth place, Hcy is viewed as a potential indicator for the presence of various cancers. Consequently, the elevation of Hcy levels due to the NNMT-catalyzed methylation of NAM may constitute a pivotal mechanism through which NNMT influences the onset and advancement of hepatocellular carcinoma, highlighting that defects in Hcy metabolism could contribute to cancer development. In an extensive genome-wide association analysis, Souto et al. [ 80 ] discovered that NNMT is a crucial genetic factor influencing the levels of Hcy in plasma. Malaguarnera et al. [ 89 ] found that, as chronic hepatitis C (HCV) progresses to cirrhosis and, ultimately, to HCC, serum folate levels gradually decreased, while plasma Hcy levels steadily increased. This phenomenon suggests that both folate and Hcy play crucial roles in disease progression and liver carcinogenesis. The reduction in folate impairs the remethylation of Hcy, leading to decreased SAM concentrations, while the plasma levels of Hcy and SAH rise. These changes collectively result in the hypomethylation of DNA and proteins, particularly histones, thereby influencing gene expression and DNA stability. Under conditions of low or deficient folate levels, plasma Hcy concentrations typically increase. Hcy, an important intermediate in the metabolism of methionine and cysteine, is elevated in a condition known as hyperhomocysteinemia (HHcy) [ 90 ]. HHcy may arise from liver disorders or impaired liver function, disrupting intracellular lipid metabolism. Moreover, cytochrome P450 (CYP) arachidonic acid epoxygenase enzymes, found in human cancers, contribute to cancer metastasis. Zhang et al. [ 91 ] investigated the interplay between CYP450 metabolism and Hcy in HCC. Their study of 42 HCC samples revealed significantly increased levels of 4-epoxyeicosatrienoic acid (EET) isomers, and the CYP2J2 enzyme responsible for their synthesis and intracellular Hcy were all significantly higher compared to corresponding non-tumor tissues. The decreased DNA methylation induced by Hcy, the essential factors for CYP2J2 expression upregulation, and EET metabolic pathway activation were the ERK1/2 signaling pathway and the SP1/AP1 binding motifs’ activation of the CYP2J2 promoter. Increased Hcy levels promoted enhanced tumor cell characteristics, a phenomenon that was reversed in vitro by silencing CYP2J2. Furthermore, in a mouse model fed 2% ( w / w ) L-methionine, with or without folate deficiency, HHcy significantly promoted tumor growth and volume, and an increased CYP2J2 expression, along with DNA demethylation patterns. Knockdown of CYP2J2 using short hairpin RNA (shRNA) markedly weakened these effects. Therefore, HHcy promotes liver cancer development by affecting the DNA demethylation of CYP2J2 and its interaction with the ERK1/2 signaling pathway, thus driving the CYP450-EET metabolic pathway, potentially contributing to the development of hepatic malignancies.

Previous research has indicated that NNMT acts as an oncogenic in liver cancer, contributing to the invasion and metastasis. Lu et al. [ 96 ] identified elevated levels of NNMT in iCCA tissues through Western blotting and immunohistochemistry (IHC). They found that NNMT levels in human iCCA tissues were markedly higher than nearby normal tissues. This finding further confirmed that increased NNMT expression boosts the invasion and metastatic potential of iCCA cells, both in vitro and in vivo. However, the expression pattern and biological role of NNMT in iCCA remain incompletely understood. The potential mechanisms underlying the crosstalk between hepatic stellate cells (HSCs) and liver cancer cells in driving HCC progression are still unclear. Li et al. [ 97 ] experimentally demonstrated that activated HSCs boost HCC’s invasive and migratory capabilities by increasing NNMT levels. They co-cultured the human liver cancer cell line SMMC-7721 with conditioned media (CM) from active or dormant HSCs, observing that the active HSC media notably enhanced HCC invasion and metastasis. Furthermore, Li et al. [ 90 ] reported that activated HSCs induce the upregulation of NNMT. The contribution of NNMT in modulating various metabolic pathways in liver cancer cells is well-understood. In HCC tissues, elevated NNMT levels are strongly linked to vascular invasion, high serum HBV-DNA, and metastatic spread. A functional analysis revealed that NNMT promotes HCC cell invasion and metastasis by altering histone H3 lysine 27 trimethylation (H3K27me3) patterns and upregulating the cluster of differentiation 44 (CD44) expression. Mu et al. [ 98 ] explored the impact of NNMT on the malignant biological behaviors of the human liver cancer cell line SMMC7721. They conducted various experiments, including MTT proliferation assays, colony formation assays, adhesion assays, and invasion assays, using SMMC7721 cells transfected with NNMT (S/NNMT), control SMMC7721 cells (S), and cells transfected with an empty vector (S/P). The MTT assay results showed no significant difference in cell proliferation or colony formation rates between the S/NNMT group and the S or S/P groups ( p > 0.05). However, the S/NNMT group exhibited a significantly higher heterotypic adhesion ability than the S and S/P groups ( p < 0.001). Transwell invasion assays showed that the S/NNMT group had a significantly higher optical density (OD) value compared to the other two groups. These results confirmed that NNMT alters the malignant traits of liver cancer cells and enhances their ability to invade and metastasize. Moreover, studies suggest that NNMT is a potential therapeutic target for the statin-induced inhibition of liver cancer cell metastasis. By promoting the Warburg effect, SIRT1 causes cancer cells to prefer glycolysis over oxidative phosphorylation for energy, even when oxygen is present—which also contributes significantly to tumor growth and invasiveness through lactate production [ 99 ]. Therefore, NNMT may enhance the Warburg effect, further supporting the invasion and metastasis of liver cancer cells. Gaining a deeper understanding of the metabolic mechanisms of NNMT might reveal new therapeutic targets for treating liver cancer.

4.1. NNMT in Liver Cancer Diagnosis: A Potential Biomarker Given the expression profile of NNMT in liver cancer tissues and serum, researchers have explored its potential application in the clinical diagnosis of HCC, suggesting that NNMT may serve as a novel tumor biomarker. Kim et al. [ 9 ] conducted an in-depth study on frozen tumor samples and nearby non-cancerous tissues from 120 primary HCC patients. They utilized the real-time reverse transcription PCR (RT-PCR) to measure the expression levels of NNMT and internal control genes, aiming to investigate the correlation between NNMT mRNA levels, clinical pathological parameters, and clinical outcomes. The results revealed that NNMT mRNA levels were significantly lower in HCC tissues compared to adjacent non-cancerous tissues ( p < 0.0001), and NNMT expression was significantly associated with the tumor stage ( p = 0.010). Further analysis showed that patients with increased levels of NNMT mRNA showed a tendency for shorter overall survival (OS) ( p = 0.053) and a marked reduction in disease-free survival (DFS) ( p = 0.016). These findings suggest that, in HCC cases, NNMT expression is closely linked to tumor stage and DFS duration. Given NNMT’s broad substrate specificity, its expression level may impact both the effectiveness and side effects of chemotherapy. Therefore, NNMT shows promise as a potential biomarker for diagnosing liver cancer and holds significant value in clinical practice.

Our study suggests that exercise can be used as a non-pharmacological strategy to modulate NNMT activity [ 131 , 132 ]. In rodent models, aerobic exercise decreases hepatic NNMT expression and improves insulin sensitivity through AMPK-SIRT1 signaling [ 133 ]. Clinical observations further suggest that patients with HCC who regularly exercise at a moderate intensity have lower serum NNMT levels and longer progression-free survival compared with sedentary patients [ 134 ]. Mechanistically, exercise enhances anti-tumor immunity by reducing immunosuppressive cells in the TME [ 135 ], which may have synergistic effects with NNMT-targeted drugs. Despite these benefits, challenges such as optimizing exercise regimens (e.g., intensity and duration) and ensuring patient compliance require further investigation.

This study delves into the central role of NNMT in driving liver cancer progression and proposes it as a promising therapeutic target for both the prevention and treatment of liver cancer. While numerous positive findings have emerged, there remain many unanswered questions regarding the precise mechanisms through which NNMT influences liver cancer and how effective clinical drugs targeting NNMT can be designed. Currently, the understanding of how NNMT influences anaerobic energy metabolism remains limited. The inhibition of NNMT has been reported to affect the bioenergetics of endothelial cells [ 137 ]. A notable characteristic of tumor cell metabolism is the abnormal increase in anaerobic glycolysis, which leads to a significant accumulation of energy derived from glucose, regardless of oxygen levels. These metabolic alterations facilitate rapid cell proliferation and tumor growth, further confirming the process of the Warburg effect [ 138 ]. Many studies stress NNMT’s key role in curbing aerobic glucose and lipid metabolism; yet, its function in anaerobic pathways remains undefined. Our previous research [ 132 ] indicated that NNMT levels are elevated in the extensor digitorum longus, which mainly depends on anaerobic metabolism, when compared to the soleus muscle, which mainly utilizes oxidative metabolism. Moreover, using MNAM to suppress NNMT has led to lower performance in anaerobic endurance experiments in rats [ 139 ]. These findings suggest that NNMT plays a significant role in modulating anaerobic metabolism, potentially influencing exercise performance, particularly in activities that demand high-intensity, anaerobic energy production. A critical gap lies in elucidating the functional heterogeneity of NNMT across tumor types. For instance, systematic comparisons between HCC and NNMT-high malignancies (e.g., breast and ovarian cancers) could delineate conserved pathways (e.g., NAD + salvage mechanisms) versus liver-specific vulnerabilities, such as the GNMT–NNMT regulatory axis in methionine cycle homeostasis. While NNMT inhibition reprograms immunosuppressive niches in ovarian cancer [ 102 ], its immunomodulatory effects on HCC-associated macrophages or stromal compartments remain unexplored. Furthermore, tissue-selective delivery strategies (e.g., hepatocyte-targeted RNAi versus pan-cancer NNMT inhibitors) may enhance therapeutic precision by balancing efficacy and toxicity profiles. Collectively, these investigations will clarify whether NNMT operates as a universal metabolic linchpin or a context-dependent orchestrator of oncogenesis. Further research is essential for evaluating the potential applicability of NNMT as a therapeutic target for liver cancer. Challenges have arisen in developing RNAi-based therapies and small-molecule NNMT inhibitors. In RNAi drug development, these challenges include issues related to safety, efficacy, and delivery systems. To overcome these hurdles, researchers have explored various delivery strategies, including viral and non-viral vectors, as well as chemical modifications, cationic liposomes, polymers, nanoparticles, and bio-conjugation methods for siRNA to enhance stability and intracellular delivery [ 140 ]. Several RNAi-based drugs, such as the aptamer pegaptanib targeting specific proteins, microRNA inhibitors like givosiran and patisiran, and antisense oligonucleotides targeting RNA, such as golodirsen and inotersen, have been approved by the U.S. Food and Drug Administration (FDA) for clinical use [ 141 ]. While RNAi therapeutics have seen considerable advancement clinically, enhancements in pharmacokinetics, pharmacodynamics, and toxicity mitigation are still needed [ 142 ]. For small-molecule inhibitors of NNMT, the challenge lies in identifying inhibitors that are both highly effective and selective, with robust activity within living organisms [ 143 ]. Currently, small-molecule NNMTi are widely used to study the pharmacological effects of NNMT, but their effectiveness is questionable due to problems with inadequate selectivity and poor metabolic stability [ 144 ]. Overall, compared to a recently published NNMT review [ 145 , 146 ], this article systematically sorted out the mechanism of NNMT regulating immune escape in liver cancer through metabolic reprogramming for the first time, and proposed its potential as a target for combination therapy. In addition, we summarize in detail the barriers to the clinical translation of small-molecule inhibitors and RNAi therapeutics for NNMT, providing a clear direction for future research.