Overexpression of NNMT in Glioma Aggravates Tumor Cell Progression: An Emerging Therapeutic Target

SIMPLE SUMMARY: Glioma is one of the most common intracranial malignancies and is incurable due to strong aggressiveness and resistance to radiotherapy and chemotherapy. The lack of effective therapeutic targets is a major problem in current treatment. In the present study, we found that nicotinamide N-methyltransferase (NNMT) is a key factor influencing the occurrence and development of glioma. High NNMT expression in glioma is a predictor of short overall survival and poor patient outcome. NNMT knockdown reduced the volume of mice xenograft glioma and the viability of glioma cells. Additionally, overexpression of NNMT epigenetically silenced GAP43 through DNA methylation, histone methylation, and deacetylation modification processes. GAP43 can inhibit the formation of microtubules in tumor and intertumor cell network connections and induce apoptosis through the SIRT1 signaling pathway. Therefore, NNMT could be a potential candidate for the clinical diagnosis and treatment of glioma. ABSTRACT: Purpose: Increasing evidence has revealed that nicotinamide N-methyltransferase (NNMT) is a key factor influencing the prognosis of tumors. The present study aimed to investigate the role of NNMT in glioma and to elucidate the associated functional mechanisms. Methods: Clinical samples were analyzed by immunohistochemical staining and Western blotting to evaluate NNMT expression in glioma and normal brain tissues. The correlation between NNMT expression and glioma was analyzed using the Cancer Genome Atlas (TCGA) database. Additionally, NNMT was knocked down in two types of glioma cells, U87 and U251, to evaluate the invasive ability of these cells. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate NNMT knockdown in the cells. Furthermore, ELISA was used to determine the balance between nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide hydrogen (NAD/NADH ratio), which verified the altered methylation patterns in the cells. The glioma xenograft mouse models were used to verify the regulatory role of NNMT, GAP43, and SIRT1. Results: Analysis based on our clinical glioma samples and TCGA database revealed that overexpression of NNMT was associated with poor prognosis of patients. Knockdown of NNMT reduced the invasive ability of glioma cells, and downregulation of its downstream protein GAP43 occurred due to altered cellular methylation caused by NNMT overexpression. Gene Set Enrichment Analysis confirmed that NNMT modulated the NAD-related signaling pathway and showed a negative association between NNMT and SIRT1. Moreover, the regulatory roles of NNMT, GAP43, and SIRT1 were confirmed in glioma xenograft mouse models. Conclusion: Overexpression of NNMT causes abnormal DNA methylation through regulation of the NAD/NADH ratio, which in turn leads to the downregulation of GAP43 and SIRT1, eventually altering the biological behavior of tumor cells.