The Role of the CTBP2 Mediated Glycolytic Pathway in Nasopharyngeal Carcinoma Metastasis

Description

Nasopharyngeal carcinoma (NPC), a highly aggressive and metastatic head and neck malignancy, exhibits invasion and metastasis closely linked to aerobic glycolysis. This study systematically analyzed the effect of CTBP2-mediated glycolytic signaling on NPC progression to elucidate the role of metabolic regulation in tumor development. Immunohistochemistry was used to assess expression and localization of CTBP2 and ZEB1 in NPC tissues, metastatic lesions, and adjacent tissues. Both proteins were highly expressed in NPC and metastatic tissues, indicating their involvement in tumorigenesis and metastasis. Statistical analysis revealed significant differences between metastatic and primary NPC tissues, with CTBP2 expression correlating with T, N, and M stages. These findings suggest that CTBP2 contributes to NPC invasion and metastasis. CTBP2 knockdown vectors were constructed, and HNE-1 cells were divided into control, si-NC, and si-CTBP2 groups. Cell viability was evaluated by CCK-8, invasion by Transwell, and metabolic alterations by extracellular acidification rate, lactate production, and glucose uptake. Compared with si-NC, si-CTBP2 significantly reduced proliferation and invasion. Glycolysis, glycolytic capacity, and reserve were decreased, while non-glycolytic acidification remained unchanged. 2-DG6P and lactate levels were reduced, indicating impaired glucose uptake and lactate generation. Collectively, CTBP2 knockdown suppressed glycolysis at the transcriptional level, reducing HNE-1 proliferation and invasion, suggesting its role in regulating glycolysis-related genes in NPC. qPCR confirmed that CTBP2 silencing also reduced ZEB1 mRNA, indicating transcriptional suppression of ZEB1. To further validate this axis, HNE-1 cells were treated with si-NC, si-CTBP2, si-CTBP2+OE-NC, and si-CTBP2+OE-ZEB1. CCK-8 and wound-healing assays showed that si-CTBP2 significantly reduced proliferation and migration, while ZEB1 overexpression reversed these effects, supporting its role in promoting NPC progression. A ZEB1 overexpression plasmid (ZEB1-pcDNA3.1(+)) was transfected into HNE-1 cells. Dual-luciferase reporter constructs (HK2-promoter-pmirGLO) were co-transfected with ZEB1-pcDNA3.1(+) into 293T cells. Overexpression significantly increased ZEB1 mRNA and enhanced HK2 promoter activity, confirming direct binding and transcriptional activation of HK2 by ZEB1, thereby facilitating glycolysis. In vivo, HNE-1 xenograft models were established in nude mice with CTBP2 knockdown or control. Tumor growth was monitored, and molecular analyses performed. CTBP2 silencing markedly inhibited tumor growth, reduced CTBP2 and ZEB1 mRNA, increased E-cadherin protein, and decreased Vimentin, GLUT1, HK2, and ZEB1 expression. In conclusion, CTBP2 regulates glycolysis and EMT in NPC through ZEB1 transcriptional control. Its silencing suppresses proliferation, invasion, and metastasis, highlighting CTBP2 as a potential therapeutic target.

Steps to reproduce

Experiment 1 Materials and Grouping: Tissue samples were collected and divided into three groups, each consisting of 10 cases: 1.Paracancerous tissue 2.Primary nasopharyngeal carcinoma (NPC) tissue 3.Metastatic tissues(lymph node) Methods: Immunohistochemical (IHC) staining was performed on paraffin-embedded pathological sections to detect the expression of CTBP2 and ZEB1. The antibodies used were ZEB1 Ab (Affinity Biosciences) and Anti-CTBP2 Rabbit pAB (Servicebio). Experimental Procedure: 1.Tissue collection: This study included 10 NPC patients with cervical lymph node metastasis. For each patient, three types of specimens were obtained: paracancerous tissue, primary NPC tissue, and etastatic tissues(lymph node). All samples were fixed in formalin, paraffin-embedded, sectioned, and histologically diagnosed by an experienced pathologist. 2.Immunohistochemistry analysis: Staining was performed in three groups (10 cases per group), with two markers (CTBP2, ZEB1). Scoring was based on staining intensity (0 = negative, 1 = weak, 2 = moderate, 3 = strong) and the percentage of positively stained cells (0 = none, 1 = <10%, 2 = 11%–50%, 3 = 51%–80%, 4 = >81%). For each slide, three random fields were selected under a 400× light microscope, and Image Pro Plus software was used to calculate the integrated optical density (IOD) and area, from which the mean density was derived. 3.Statistical analysis: Data were analyzed using SPSS 25.0 software. Independent sample t-tests and rank-sum tests were used for pairwise comparisons, and one-way ANOVA and rank-sum tests were applied for multiple group comparisons. A p-value <0.05 was considered statistically significant. 4.Reproducibility: Each patient provided three specimens forming one complete set, and a total of 10 sets were analyzed. Experiment 2 Materials and Grouping: Three experimental groups were established: 1.Control group 2.si-NC (negative control) 3.si-CTBP2 Methods: Human nasopharyngeal carcinoma cell lines (HNE-1, C666-1, HK-1) and normal nasopharyngeal epithelial cells (NP69) were cultured. Baseline expression of CTBP2 was evaluated, and HNE-1 cells were selected for subsequent experiments.Following validation, cell viability was assessed using CCK-8 assays, while wound healing and Transwell assays were performed to evaluate cell migration and invasion. Lactate production, glucose uptake, and extracellular acidification rate were measured using commercial assay kits. qPCR was used to detect CTBP2 and ZEB1 expression, while Western blotting was employed to assess E-cadherin, vimentin, GLUT1, HK2,

Institutions

• Jinan University

  Tripoli

Categories

Funders

• Shenzhen Science and Technology Innovation Commission

  Guangdong, China

  Grant ID: JCYJ20230807141514029

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