Abstract Glioma, the most prevalent primary brain tumor in adults, is characterized by significant invasiveness and resistance. Current glioma treatments include surgery, radiation, chemotherapy, and targeted therapy, but these methods often fail to eliminate the tumor completely, leading to recurrence and poor prognosis. Immune checkpoint inhibitors, a class of commonly used immunotherapeutic drugs, have demonstrated excellent efficacy in treating various solid malignancies. Recent research has indicated that unconventional levels of expression of the MAP2K3 gene closely correlates with glioma malignancy, hinting it could be a potential immunotherapy target. Our study unveiled substantial involvement of MAP2K3 in gliomas, indicating the potential of the enzyme to serve as a prognostic biomarker related to immunity. Through the regulation of the infiltration of immune cells, MAP2K3 can affect the prognosis of patients with glioma. These discoveries establish a theoretical foundation for exploring the biological mechanisms underlying MAP2K3 and its potential applications in glioma treatment. Keywords: glioma, immunotherapy, MAP2K3, tumor immune microenvironment, prognosis Background Glioma is the most common primary brain tumor and is a general term for a large group of intracranial primary tumors that occur from glial cells derived from neural ectoderm. Gliomas can be categorized as grade I–IV according to the World Health Organization (WHO) grading standards. Glioblastoma (GBM) is a high-grade glioma, defined as WHO grade IV, and is one of the most lethal gliomas. It makes up 70–75% of all diffuse gliomas, and GBM patients have a median survival time of about 14–17 months. The main treatment options are surgery, radiotherapy, temozolomide (TMZ) chemotherapy and radiotherapy combined with TMZ chemotherapy, but none of the treatment outcomes are satisfactory ([35]1). The surgical elimination of the tumor, followed by chemotherapy and radiation therapy, is the conventional treatment for glioblastoma. As a result of the extremely aggressive characteristics of glioma cells, complete removal of the tumor is currently difficult to achieve ([36]2). The potential mechanisms of glioma migratory invasion remain to be further investigated ([37]3). Immunotherapy is able to inhibit tumor growth and spread by activating the patient’s own immune system. Common immune cell types in glioma patients include T cells, B cells, dendritic cells, and natural killer cells; which are closely related to tumor immune escape and drug resistance ([38]4). Immunotherapy has been demonstrated to improve patient survival as well as quality of life in glioma patients ([39]5). Additionally, immunotherapy may be used together with more established treatments like radiation and chemotherapy to maximize therapeutic outcomes. Despite the clinical effectiveness of immunotherapy in the management of gliomas, additional investigation is required to address potential issues with immune escape and drug resistance ([40]5). Mitogen-activated protein kinase 3 (MAP2K3) is a member of the bispecific protein kinase kinases (MKK) group, which is found in the Mitogen-activated protein kinase pathway (MAPK) ([41]6). The MAP2K3 protein was first discovered in 1996, and current research has focused on its role as an activator of the p38-MAPK signaling pathway ([42]7). In the therapeutic studies of esophageal squamous cell carcinoma, MAP2K3 inhibitors have been reported to be effective in inhibiting cell growth. MAP2K3 can mediate cellular responses to external stimuli by phosphorylating and activating the p38-MAPK signaling pathway ([43]8). When cells are subjected to external stimuli, activated MAP3K activates MAP2K3 to phosphorylate and activate the p38-MAPK signaling pathway. The downstream target proteins that are regulated by the active p38-MAPK signaling pathway are also regulating additional biological processes such as cell proliferation, differentiation, and apoptosis. p38-MAPK family proteins play complex and diverse roles in tumors ([44]9, [45]10). In order to exert an anti-tumor effect, p38-MAPK activation may induce apoptosis and cell cycle arrest in a tumor cell ([46]11). In addition, it has been shown that the p38-MAPK protein can regulate activity of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the phosphoinositide 3-kinase/Akt (PI3K/AKT) signaling pathways, which in turn promotes tumor cell proliferation and growth ([47]12). The p38-MAPK signaling pathway may promote tumor growth by regulating inflammatory responses and angiogenesis in the tumor microenvironment ([48]11). Additionally, this pathway contributes to immunomodulation in the tumor microenvironment. The p38-MAPK signaling pathway can support immunological functions like tumor immune surveillance and immune antigen presentation by modulating the activity of immune cells and the production of immune components ([49]13). p38-MAPK signaling pathway activation can promote the production and secretion of cytokines, such as interferon gamma (IFN-γ), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and interleukin 1 (IL-1), which can promote the activation of immune cells and an immune response ([50]6). In addition, the p38-MAPK signaling pathway can also regulate the activity of antigen-presenting cells, such as macrophages, to enhance the immune response ([51]6). Therefore, a thorough investigation of the function of the MAP2K3 gene, which is closely associated with the p38-MAPK signaling pathway, in the immune microenvironment of glioma tumors may be helpful in understanding the mechanisms underlying glioma development and growth, and result in the development of novel therapeutic methods and targets. In our study, MAP2K3 expression was discovered to be aberrantly high in a range of tumor tissues, and such high expression was found to be associated with poor clinicopathological characteristics and outcome of gliomas. We found that the genes related to MAP2K3 were primarily enriched in immunomodulatory pathways through functional and pathway enrichment analysis. Finally, we discovered a relationship between MAP2K3 expression and immunological checkpoints, immune-related genes, and immune infiltration in glioma. Taken together, our research highlights the critical function of MAP2K3 in tumor immune modulation and glioma prognosis; indicating the MAP2K3 gene as a potential novel target for the treatment of glioma (see [52]Table 1). Table 1. Summary of the relevant databases. Name of database Link CGGA [53]http://www.cgga.org.cn/ CancerSEA [54]http://biocc.hrbmu.edu.cn/CancerSEA/ TIMRE [55]https://cistrome.shinyapps.io/timer/ GEPIA website [56]http://gepia.cancer-pku.cn/index.html Ivy Glioblastoma Atlas Project [57]https://glioblastoma.alleninstitute.org/ UCSC [58]https://xenabrowser.net/datapage/ TIGER [59]http://tiger.canceromics.org/ The Human Protein Atlas [60]https://www.proteinatlas.org TCGA [61]https://cancergenome.nih.gov/ TISCH ([62]14) [63]http://tisch.comp-genomics.org/home/ [64]Open in a new tab Materials and methods Collection of data and analysis of MAP2K3 expression In this study, clinically relevant mRNA expression profile datasets were obtained from the public databases the Gene Expression Omnibus (GEO), the Chinese Glioma Genome Atlas (CGGA), and The Cancer Genome Atlas (TCGA). We utilized the R software for the initial processing of gene expression profiles, which encompassed tasks such as background correction, normalization, and Log 2 transformation. Subsequently, to evaluate the presence of MAP2K3 in gliomas and various other tumors, we used the TIMRE database. This allowed us to investigate MAP2K3 expression levels in tumor samples, juxtaposed with their corresponding healthy tissues. Using the GEPIA (Gene Expression Profiling Interactive Analysis) website, we also investigated the expression of MAP2K3 in low grade glioma (LGG), GBM, and normal tissues. The expression of MAP2K3 in gliomas of different WHO classifications was examined. Using the Human Protein Atlas website, we evaluated the amounts of MAP2K3 protein expression in glioma and normal brain tissues as well as the location of MAP2K3 protein in glioma cells. Analysis of MAP2K3 protein expression levels in gliomas We used immunohistochemistry to evaluate the differential expression levels of MAP2K3 protein in glioma tissue compared to normal brain tissue. Images from immunohistochemical staining of normal brain tissue, LGG, and HGG were separately sourced from The Human Protein Atlas database, specifically the cerebral cortex section. The antibody used for immunohistochemistry was anti-MAP2K3 primary antibody (HPA043783). Evaluation of MAP2K3’s prognostic significance in glioma In this study, both univariate and multivariate Cox regression analyses were performed to determine whether MAP2K3 might be employed as an independent prognostic factor for glioma. WHO categorization, IDH status, gender, 1p/19q code, and age were clinical variables included in the Cox regression analysis ([65]15). The R package “rms” was used to generate column line plots and calibration. To predict overall survival (OS) at 1, 3, and 5 years, we used the “survivor” package. We utilized the “pROC” R tool to generate AUC curves for the ROC study. We additionally investigated the relationship between MAP2K3 and Overall Survival (OS) and Progress Free Survival (PFS) in several clinical cohorts with LGG and GBM. Gene set variation analysis In Gene Set Variation Analysis (GSVA), the distribution of genes in a predefined set of genes is used to assess their trends in a table of phenotypically related ordered genes to determine their role in phenotype definition. To investigate the biological significance of MAP2K3, the “GSVA” package in R was used in this study to perform GSVA analysis. Based on their mRNA expression levels, MAP2K3 was split into low and high expression groups to identify the functional and pathway significance differences between the two groups. We downloaded “h.all.v7.2.symbols” and “c2.cp.kegg.v7.2.symbols” from the MsigDB database as reference gene sets for GMT (Hallmarks) and KEGG pathways, respectively. The “Limma” program was used to analyze the differences in GSVA pathways between patients in the high and low MAP2K3 groups, with adjusted criteria of p < 0.05 and abs (log2FC) > 0.3. Through heat maps, we displayed the Hallmarks and KEGG differential pathways individually. Evaluation of immunological microenvironment and tumor immune cell infiltration In order to figure out immune, stromal, and ESTIMATE scores; our study first examined immune and stromal cell types according to gene expression profiling using the “ESTIMATE” R package. Then, we assessed the correlation between the MAP2K3 gene and various immune cell levels using the CIBERSORT and ssGSEA algorithms, and discovered a link between the level of MAP2K3 expression and the infiltration of various immune cell types. Evaluation of immunotherapy-related predictors In this study, we compared the expression of several immunological checkpoints in the groups with high and low levels of MAP2K3 expression. The Wilcoxon rank sum test was used to evaluate the differences in immune checkpoint expression between the high MAP2K3-expressing and low MAP2K3-expressing groups. In order to demonstrate the sensitivity of the relevant subgroups to immune checkpoint inhibitor (ICI) therapy, we calculated the Tumor Immune Dysfunction and Exclusion TIDE (TIDE) scores for the high MAP2K3 expression group and the low MAP2K3 expression group. TIDE scores are used to assess the effectiveness of immunotherapy, with high TIDE scores indicating high tumor tolerance to immune checkpoint inhibitor therapy and low TIDE scores indicating better treatment outcomes. We then calculated the interferon gamma (IFNG) score, T cell receptor abundance (TCR), TCR Shannon score, microsatellite instability (MSI) and single nucleotide variant (SNV) neoantigens from TCGA. These metrics can be used to predict the ability of T cells in the immune microenvironment to exert tumor suppression and calculate levels of tumor neoantigens. Somatic cell mutation analysis In this study, somatic mutations and copy number alterations (CNAs) were downloaded from the TCGA database, and VarScan2 software was used to whole-genome sequence data of somatic mutations in the high MAP2K3 expression group and low MAP2K3 expression group. The Fisher’s exact test was used to discover various mutation patterns, the CoMEt algorithm was utilized to find both co-occurring and mutated genes, and p < 0.05 was established as the threshold for choosing differentially mutated genes. For the purpose of visualizing somatic mutations, “maftools” was a R package. Single-cell sequencing to assess MAP2K3 expression levels in gliomas CancerSEA and TISCH, two single-cell sequencing data platforms, were employed to evaluate MAP2K3 expression at the single-cell level in gliomas. In vitro validation of MAP2K3’s function in glioma U251 glioma cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, maintained at 37°C with 5% CO[2]. For gene silencing, cells underwent transfection with siRNA targeting MAP2K3 and a control siRNA from GenePharma (Shanghai, China), using Lipofectamine 3,000 as per manufacturer’s guidelines. MAP2K3 knockdown was verified by qRT-PCR 48 h post-transfection using specific primers ([66]16). The primers used were as follows: for MAP2K3; Forward: GACTCCCGGACCTTCATCAC, Reverse: GGCCCAGTTCTGAGATGGT, and for GAPDH; Forward: TGTGGGCATCAATGGATTTGG, Reverse: ACACCATGTATTCCGGGTCAAT. The CCK-8 kit was deployed to ascertain the viability of U251 cells, as well as the survival of U251 cells. Wound healing assay Cellular migration was assessed using a wound healing assay. Cells seeded in 6-well plates were grown to confluence, and a sterile 200 μL pipette tip was used to scratch a line through the monolayer. After washing away debris with phosphate-buffered saline, the cells were incubated in serum-free medium. Images were captured at 0 and 24 h post-scratch using an inverted microscope, and the rate of migration was quantified by measuring the gap with ImageJ software. Transwell migration assay For the Transwell migration assay, cells were suspended in serum-free medium (1 × 10^5 cells/mL) and 100 μL was placed into the upper chamber of a Transwell insert (Corning, United States). The lower chamber contained 600 μL of medium with 10% fetal bovine serum as a chemoattractant. Following a 24 h incubation at 37°C and 5% CO[2], cells on the upper membrane were removed, while those on the lower surface were fixed, stained with crystal violet, and counted in five fields under a light microscope. Statistical analysis The Wilcoxon test was used in this study to compare the differences in MAP2K3 expression between normal tissues and gliomas in the dataset obtained from the GEO database. We compared the variations in MAP2K3 expression in gliomas of various WHO classifications using the data acquired from TCGA and the Kruskal–Wallis test. To examine the association between survival and MAP2K3 expression levels, Kaplan–Meier curves were used. Results MAP2K3 is differentially expressed in gliomas and multiple other tumors To explore the expression pattern of MAP2K3 in gliomas, we first analyzed the expression of MAP2K3 in tumor tissues. We used the TIMER database to explore the expression of MAP2K3 in 33 human cancers and found that MAP2K3 was expressed in multiple tumors ([67]Figure 1A). The HR values of the MAP2K3 gene in LGG and GBM were higher than 1, suggesting that high MAP2K3 expression is associated with an increased risk of LGG and GBM occurrence ([68]Figure 1D). We also used the GEPIA2 website to examine the TCGA database in order to investigate the expression of MAP2K3 in gliomas and healthy brain tissues. The findings demonstrated that GBM had higher levels of MAP2K3 mRNA expression than normal brain ([69]Figure 1B). In addition, the expression level of MAP2K3 in gliomas correlated with the WHO grade of glioma, and the expression level of MAP2K3 in gliomas increased with the grade of glioma ([70]Figure 1C). We found higher levels of MAP2K3 expression in multiple glioma cohorts with WHO grade 3 gliomas than WHO grade 2 ([71]Figures 1G–[72]K). MAP2K3 expression levels were also upregulated in the single-cell EXP0059 glioma cell group ([73]Figures 1E,[74]F). Figure 1. [75]Figure 1 [76]Open in a new tab MAP2K3 is differentially expressed in gliomas. (A) MAP2K3 is differentially expressed in pan-cancer (*p < 0.05, **p < 0.01, and ***p < 0.001, Wilcoxon test). (B) MAP2K3 is differentially expressed between GBM and normal patients (*p < 0.05, **p < 0.01, and ***p < 0.001, Wilcoxon test). (C) MAP2K3 is differentially expressed among three WHO grades (*p < 0.05, **p < 0.01, and ***p < 0.001, Wilcoxon test). (D) Prognostic significance of MAP2K3 in pan-cancer. (E) MAP2K3 is highly expressed compared with housekeeping genes. The box plot illustrates the distribution of MAP2K3 gene expression in the glioma EXP0059 dataset. (F) The t-SNE plot showed the expression distribution of MAP2K3. T-SNE describes the distribution of cells, every point represents a single cell, and the color of the point represents the expression level of MAP2K3 in the cell. (G–K) MAP2K3 is differentially expressed in CGGA301 (G), CGGA325 (H), CGGA693 (I), [77]GSE108474 (J), and TCGA-LGG (K) cohorts. (L) Representative images of MAP2K3 among different grades in immunohistochemistry. (M) Immunofluorescence staining of MAP2K3 in the SH-SY5Y cell line. We then assessed the protein expression level of MAP2K3 in gliomas using The Human Protein Atlas database. According to the results of immunohistochemical staining, glioma tissues generally express more MAP2K3 than healthy brain tissues do, and high-grade gliomas express more of the MAP2K3 protein ([78]Figure 1L). We evaluated the localization of MAP2K3 protein in the glioma cell line SH-SY5Y, and the results showed that MAP2K3 was localized in the cytoplasm ([79]Figure 1M). These results show that MAP2K3 is substantially expressed in both high-grade and low-grade gliomas, and that its expression level rises with increasing WHO grades. This suggests a potential association between MAP2K3 and the malignant behavior of gliomas. Patient prognosis is correlated with MAP2K3 expression in gliomas By analyzing multiple GBM cohorts and LGG cohorts, we found that MAP2K3 expression levels differed among glioma patients by age, 1p/19q co-deletion and gender; with higher MAP2K3 expression levels in young and middle-aged (<60 years), 1p/19q non-co-del, and male glioma patients ([80]Figures 2A–[81]K). Figure 2. [82]Figure 2 [83]Open in a new tab Differential expression of MAP2K3 in different clinical features. (A–D) MAP2K3 is differentially expressed between different ages in TCGA-GBM (A), CGGA301 (B), CGGA325 (C), and TCGA-LGG (D) cohorts. (E–J) MAP2K3 is differentially expressed between different 1p19q status in CGGA301 (E), CGGA325 (F), CGGA693 (G), E-MTAB-3892 (H), CGGA325 (I), and CGGA693 (J) cohorts. (K) MAP2K3 is differentially expressed between different genders in [84]GSE61335 cohort (*p < 0.05, **p < 0.01, and ***p < 0.001, Wilcoxon test). To investigate whether high expression of MAP2K3 could be an independent predictor of glioma prognosis, univariate and multivariate Cox regression analyses were conducted. Univariate Cox regression analysis showed that MAP2K3 expression, WHO staging, and age were associated with the prognosis of glioma ([85]Figure 3A). Multivariate Cox regression analysis revealed that MAP2K3 expression, WHO staging, and age were independent prognostic factors affecting glioma prognosis ([86]Figure 3B). Furthermore, by performing Cox regression analysis on multiple GBM cohorts and multiple LGG cohorts, we found that the MAP2K3 gene was a significant risk factor for poor patient prognosis ([87]Figures 3C,[88]D). Figure 3. [89]Figure 3 [90]Open in a new tab Prognostic significance of MAP2K3 in multi-center cohorts. (A) Univariate Cox regression analysis of Stage, age, and MAP2K3 expression. (B) Multivariate Cox regression analysis of Stage, age, and MAP2K3 expression. (C) Univariate Cox regression analysis of MAP2K3 expression in multi-center GBM cohorts. (D) Univariate Cox regression analysis of MAP2K3 expression in multi-center LGG cohorts. We discovered through a multiple cohort survival study that patients with high expression levels of MAP2K3 had shorter overall survival, regardless of whether they had high- ([91]Figures 4A–[92]I) or low-grade gliomas ([93]Figures 4J–[94]N). The Nomogram and calibration curves demonstrate that MAP2K3 is an independent prognostic factor that accurately predict patient prognosis at 1, 3, and 5 years; indicating that MAP2K3 is a good predictor of prognosis for glioma patients in the multiple regression model ([95]Figure 4O). Time-dependent analysis of ROC showed AUC values of 0.89, 0.92, and 0.92 for glioma at 1, 3, and 5 years, respectively, indicating a high predictive power ([96]Figure 4P). As a result, MAP2K3 may be employed as a glioma diagnostic marker. These results all point to MAP2K3’s prognostic potential in gliomas. Figure 4. [97]Figure 4 [98]Open in a new tab Survival analysis of expression level of MAP2K3. (A–I) OS Kaplan–Meier survival curves between glioma patients with high and low expression level of MAP2K3 in multi-center GBM cohorts. (J–N) OS Kaplan–Meier survival curves between glioma patients with high and low expression level of MAP2K3 in multi-center LGG cohorts. (O) Plots depicted the calibration of the nomogram. (P) Predictive accuracy at 1, 3, and 5 years of the nomogram in TCGA cohort. (Q) The nomogram plot revealed the prognostic prediction model based on MAP2K3, stage, and age in TCGA cohort. The potential biological mechanism of MAP2K3 in glioma To investigate the potential biological mechanisms of MAP2K3 in gliomas, we explored the function of MAP2K3 molecules in multiple cancer-related signaling pathways in the TCGA cohort. To analyze Hallmarker pathway differences between gliomas with two different MAP2K3 expression levels ([99]17), we performed GSVA gene enrichment analysis. “Inflammatory response,” “interferon gamma response,” “NF-κB/TNFA signaling pathway,” “complement,” “IL6/JAK/STAT3 signaling pathway,” and “interferon α response,” which are vital for inflammatory and immunological responses, were significantly elevated. This suggests that high MAP2K3 expression levels are closely related to immune-related signaling pathways ([100]Figure 5A). To further explore the biological pathways of gliomas at both MAP2K3 expression levels, we performed KEGG enrichment analysis on the high MAP2K3-expressing and low MAP2K3-expressing groups of the TCGA cohort. We discovered that the group with high MAP2K3 expression was primarily related to “autoimmune thyroid disease,” “IgA-producing intestinal immune network,” “systemic lupus erythematosus,” and “antigen processing and presentation.” The MAP2K3 expression group was mainly associated with “autoimmune thyroid disease,” “IgA producing intestinal immune network,” “systemic lupus erythematosus,” “antigen processing and presentation” and other processes related to immune response ([101]Figure 5B). These findings all point to MAP2K3’s potential involvement in immune-related pathways in gliomas. Figure 5. [102]Figure 5 [103]Open in a new tab Investigations of MAP2K3-related signal pathways. (A,B) GSVA enrichment analyses between low and high MAP2K3 expression group illustrated the activation status of Hallmark (A) and KEGG (B) pathways in TCGA cohort. Pink and blue represent activation and inhibition of the pathway, respectively. (C) The Wilcoxon rank-sum test revealed the variances in the normalized scores of ten cancer-related signaling pathways between the low and high MAP2K3 expression group (*p < 0.05, **p < 0.01, and ***p < 0.001). Based on previous publications, we performed ssGSEA enrichment scoring of 10 classical oncogenic signaling pathways for two MAP2K3 expression levels in TCGA_GBM and LGG cohorts. Scoring signaling pathways including Wnt, TP53, TGF, RAS, PI3K, NRF2, NOTCH, MYC, cell cycle, and Hippo pathways. Based on the enrichment analysis outcomes, groups with higher MAP2K3 expression demonstrated elevated scores in several signaling pathways; namely the Hippo, NRF2, PI3K, and TGF pathways ([104]Figures 5C,[105]D). Each of these pathways are known to be closely intertwined with tumor immune evasion responses ([106]18–22). MAP2K3-associated somatic mutations in glioma We analyzed somatic mutations in glioma patients from the TCGA cohort to investigate the mechanisms associated with MAP2K3 expression levels. Non-synonymous mutations are mutations that result in altered amino acid sequences ([107]23). Some nonsynonymous mutations lead to mutations in tumor-associated genes, which may result in enhanced cell proliferation and invasiveness. Synonymous mutations are mutations in which genomic variants do not lead to amino acid sequence alterations ([108]24). Although synonymous mutations do not directly alter the structure and function of proteins, they may affect the expression level and regulation of proteins. These aberrantly expressed proteins or peptides can be recognized by the immune system as allosteric antigens, triggering an immune response. Many tumor somatic mutations can be targets for immunotherapy and thus improve the therapeutic effect. Studying mutations in tumor cells and uncovering mutation-related molecular mechanisms can provide important references