IGF2BP3-mediated regulation of GLS and GLUD1 gene expression promotes treg-induced immune escape in human cervical cancer

Abstract

This study aimed to investigate the impact of IGF2BP3, a well-known m6A modification-related protein, on the metabolic and immune microenvironment of human cervical cancer. Bioinformatics analysis was performed to analyze the expression of IGF2BP3 in various databases, and its findings were validated using human cervical cancer tissue microarrays. We conducted a study to investigate the impact of IGF2BP3 on glutamine metabolism in cervical cancer cells through the application of metabolomics and metabolic flow analysis. Additionally, we explored how cervical cancer cells promote immune escape by secreting glutamine-derived lactate in a 3D culture setting. To identify the specific targets of IGF2BP3 that influence glutamine metabolism in cervical cancer, we employed RIP-seq analysis. IGF2BP3 exhibited high expression levels in multiple cervical cancer datasets, and its expression was significantly associated with the prognosis of cervical cancer patients. In mixed 3D cell cultures of cervical cancer and T cells, IGF2BP3 was found to enhance glutamate and glutamine metabolism in cervical cancer cells by up regulating the expression of GLS and GLUD1 genes. Moreover, it influenced the differentiation of Treg cells by promoting lactate production and secretion in cervical cancer, leading to immune escape. Mechanistic analysis revealed that IGF2BP3 stabilized the mRNA of GLS and GLUD1 genes through m6A modification, thereby facilitating glutamate and glutamine metabolism in cervical cancer cells and regulating lactate production. Additionally, we investigated the correlation between GLS, GLUD1 protein expression, and IGF2BP3 expression in human cervical cancer through multicolor immunofluorescence staining. The relevance of IGF2BP3 in the context of Treg cell-associated immune escape in cervical cancer was also confirmed. IGF2BP3 exhibits high expression in human cervical cancer and plays a crucial role in stabilizing the mRNA of GLS and GLUD1 genes, key metabolic enzymes in glutamate and glutamine metabolism, through m6A modification. This process leads to immune escape in cervical cancer by promoting lactate production and secretion.

Keywords: IGF2BP3; M6A modification; cervical cancer; glutamine metabolism.

Figure 1

IGF2BP3, a gene involved in M6A RNA modification, exhibits high expression in human cervical cancer tissues. A: Volcano plot depicting the expression levels of differentially expressed RNA-binding proteins in tumor and normal tissues, based on RNA-seq data from TCGA and GEO datasets associated with human cervical cancer. B: Venn diagram illustrating the overlapping differential RNA-binding protein genes identified in the TCGA and GEO datasets. C: Comparative analysis of IGF2BP3 gene expression between cancerous and paracancerous tissues using data from the TCGA database. D: Immunohistochemical staining (X100 for upper panel, and X400 for lower panel) showing the protein expression of IGF2BP3 in human cervical cancer tissues. E: Survival analysis conducted for patients with cervical cancer based on the expression levels of IGF2BP3. F, G: IGF2BP3 protein expression in normal tissues, hyperplasia, and different stages of cervical cancer tissues. All experiments were performed in triplicate, and statistical significance was considered at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

IGF2BP3 plays a role in promoting glutamate and glutamine metabolism in cervical cancer cells by enhancing the transcription of GLS and GLUD1 gene. A: Volcano plot displaying differential gene expression in RNA-seq analysis of wild-type and IGF2BP3 knockdown cervical cancer cells. B, C: Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses conducted using the differentially expressed genes. D: Heat map illustrating the results of gene set variation analysis (GSVA) based on RNA-seq data from wild-type and IGF2BP3 knockdown cervical cancer cells. E: Heat map presenting the differential expression of genes associated with the glutamine gene set. F: Western blot assay depicting the protein expression of IGF2BP3 in wild-type and IGF2BP3 knockdown cervical cancer cells, with α-Tubulin serving as an internal reference gene. G: Real-time PCR analysis showing the transcript levels of IGF2BP3, GLS, and GLUD1 genes in wild-type and IGF2BP3 knockdown cervical cancer cells. All experiments were performed in triplicate, and statistical significance was considered at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

IGF2BP3 plays a role in promoting glutamate and glutamine metabolism in cervical cancer cells. A: Heat map illustrating the levels of differentially expressed metabolites in wild-type and IGF2BP3 knockdown cervical cancer cell lines. B: Enrichment analysis using differential metabolites through Metabolite Set Enrichment Analysis (MSEA). C: Measurement the levels of glutamate, α-ketoglutarate, and lactate in wild-type and IGF2BP3 knockdown cervical cancer cell lines. D: IGF2BP3, GLS and GLUD1 protein expression detected by using western-blot in cervial cancer cell lines treated indicated in the figure. E: Measurement the levels of glutamate, α-ketoglutarate, and lactate in cervial cancer cell lines treated indicated in the figure. F: Measurement the levels of glutamate, α-ketoglutarate, and lactate in Caski cells treated indicated in the figure. All experiments were performed in triplicate, and statistical significance was considered at P < 0.05.

Figure 4

IGF2BP3 plays a role in promoting glutamate and glutamine metabolism in cervical cancer cells. A: Schematic model of glutamine metabolism in cancer cells. Red circles represent carbons derived from [U-¹³ C5] glutamine, and hollow circles are unlabeled. The black arrows indicate oxidative carboxylation flux from glutamine. B: Mass isotopologue distributions of glutamate in TCA cycle metabolites including α-KG, succinate, fumarate, malate, pyruvate and lactate in Caski cells treated with the control siRNA or IGF2BP3 siRNAs. Cells were cultured in [U-¹³ C5] glutamine for 24 h before metabolites extraction and GC-MS analysis; n=6, data in d-h are shown as the mean ± SD. statistical significance was considered at P < 0.05.

Figure 5

IGF2BP3 facilitates the differentiation of Treg cells by enhancing lactate production through the promotion of glutamine metabolism. A: Gating strategy employed for the detection of Treg cells in 3D cell spheres. B, C: Flow cytometry analysis depicting the impact of various treatments on the proportion of Treg cells in 3D cell spheres, as illustrated in the Figure. D, E: Immunofluorescence staining (×200) showcasing the influence of different treatments on the proportion of Treg cells in 3D cell spheres. F: Lactate determination of supernatant of cell culture treated differently indicated in the figure. All experiments were performed in triplicate, and statistical significance was considered at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

IGF2BP3 regulates the expression of GLS and GLUD1 in an m6A-dependent mechanism. A: Integrative Genomics Viewer (IGV) tracks of m6A and IGF2BP3 RIP-seq of GLUD1 and GLS. B, C: Real-time PCR analysis depicting the expression levels of IGF2BP3 and the enrichment of GLS and GLUD1 gene mRNA through m6A RIP-seq. D: TCGA-CESC dataset demonstrating a linear correlation between IGF2BP3 and the genes GLUD1 and GLS. E, F: Evaluation of the impact of IGF2BP3 loss on the stability of GLUD1 and GLS in HL-60 and KG-1 cells. Transfected cells were treated with 5 µg/ml actinomycin D for 0 h, 3 h, or 6 h prior to RNA extraction. All experiments were performed in triplicate, and statistical significance was considered at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

IGF2BP3 regulation of GLS and GLUD1 expression and promotes Treg-induced immune escape in human cervical cancer. A: Multicolor immunostaining illustrating the protein expression of IGF2BP3, GLS, GLUD1, CD4, and Foxp3 in human cervical cancer tissues (× 100 for main figure, × 200 for enlarged figure). B: Linear correlation analysis demonstrating the relationship between GLUD1 and GLS protein expression levels and IGF2BP3 in human cervical cancer tissues. C: Comparison of GLUD1 and GLS protein expression in cervical cancer tissues with high and low levels of IGF2BP3. D: Linear correlation analysis between the number of CD4+ T cells, Treg cells, and the protein expression levels of IGF2BP3 in human cervical cancer tissues. E: Comparison of the number of CD4+ T cells and Treg cells in cervical cancer tissues with high and low expression levels of IGF2BP3. All experiments were performed in triplicate, and statistical significance was considered at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.