Targeting EHMT2 inhibition in glioblastoma: effects on tumor progression and STAT3 signaling

Introduction

Glioma is the most common primary malignant tumor of the nervous system. In recent years, with the progress of science and technology and the further promotion of microsurgery, most glioma can be removed entirely, supplemented by postoperative radiation, chemotherapy and other auxiliary treatments such as gene therapy, immunotherapy, electric field, and targeted drug therapy. However, the prognosis of glioma patients still shows no noticeable improvement; the median survival of glioblastoma (GBM) patients is only 15 to 18 months (1). Therefore, the development of new and more effective targeted therapies is required.

Histone methylation is regulated by specific histone methyltransferases and demethylases and is a reversible dynamic process. Therefore, the abnormal expression of histone methyltransferases and demethylases are important in malignant tumors, especially glioma. Studies have demonstrated that in glioma cells, a variety of methyltransferases undergo significant changes (2). As an essential methyltransferase, EHMT2 (G9a) is also vital for the epigenetic regulation of cell growth. G9a (EHMT2) and proteins closely related to G9a (GLP, EHMT1) can methylate and inactivate the tumor suppressor gene TP53 (3). Elevated levels of EHMT2 have been observed in various malignant tumors, such as liver cancer, prostate cancer, lung cancer, and leukemia, while tumor growth has been shown to be significantly inhibited after EHMT2 knockout (3-6). These results suggest that histone methylation mediated by EHMT2 is closely related to tumorigenesis and development. Meanwhile, EHMT2 can be considered as a new anti-tumor target worthy of further study.

BIX-01294 is an artificial inhibitor of EHMT2, which Kubicek et al. discovered through a high-throughput screening of 125,000 compounds and has high selectivity (6). A classical study demonstrated that treatment with BIX-01294 reduced the methylation activity of EHMT2 and H3K9, which resulted in decreased chromosomal stability and further increased apoptosis and cycle arrest in malignant tumors (7). Meanwhile, in bladder carcinoma, BIX-01294 regulates tumor growth by mediating the AMPK/mTOR signaling pathway to promote autophagy (8).

In previous studies on glioma, EHMT2 and its inhibitor BIX-01294 have not been extensively studied. This study aimed to explore the effects of BIX-01294 on the proliferation, invasion, and self-renewal of GBM cells by studying EHMT2 and BIX-01294. Furthermore, the relationship between EHMT2 and cancer-associated signaling pathways, especially the signal transducer and activator of transcription 3 (STAT3) pathway, was evaluated. We intend to create a research foundation for the comprehensive understanding of the role of EHMT2 in GBM cells and the efficacy of BIX-01294 therapy in GBM. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0235/rc).

Methods

Cell culture

GBM cell lines U87 and SHG140 were obtained from Cell Bank of the Chinese Academy of Sciences and Typical Culture Collection Center. We confirm the STR analysis and mycoplasma detection of U87 and SHG140 cell. The cells were cultured in 10% FBS and 1% penicillin-streptomycin in a 5% CO₂, humidified atmosphere at 37 ℃. U87 and SHG140 cells were treated with magnetic activated cell sorting (MACS), for stem cell culture, CD133⁺ cells were grown in DMEM/F12 medium supplemented with 10 ng/mL EGF, 10 ng/mL bFGF, and B27 (1:50, Invitrogen, California, USA). The CD133⁺ neurospheres named U87s (U87 stem cell) and SHG140s (SHG140 stem cell) can be observed on the second day.

Data collection and analysis

Transcriptomic data from RNA sequencing (RNA-seq) and clinical information on glioma were mainly sourced from The Cancer Genome Atlas (TCGA, https://cancergenome.nih.gov/) and the Chinese Glioma Genome Atlas (CGGA, https://www.cgga.org.cn/). We utilized R programming (version 4.4.2) to visualize the results of the relevant statistics, which included bar charts displaying differential gene information, prognostic survival curves, heat maps, and correlation analysis plots. The identified mRNA genes were inputted into the protein-protein interaction (PPI) network via the STRING database (https://string-db.org/), with a confidence score threshold set above 0.8 for processing. Subsequently, we visualized the PPI network using Cytoscape (version 3.8.1). The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Western blotting and immunofluorescence

For Western blot analysis, we prepared RIPA buffer (a mixture of protease and phosphatase inhibitors at a concentration of 1%) and extracted total protein from the cells at a low temperature. We measured the protein concentration using the standard BCA assay (Sigma, Catalog Number QPBCA). After processing, protein samples underwent electrophoresis, with careful voltage adjustments, followed by the transfer to polyvinylidene fluoride (PVDF) membranes. To prevent nonspecific binding, we blocked the PVDF membranes with 5% Tris-Buffered Saline with Tween-20 (TBST) containing nonfat milk for about half an hour, then washed the membranes three times with PBST on a shaker. The target protein primary antibody was incubated on a shaker at 4 ℃ overnight. Following the recovery of the primary antibody, we washed the membranes three times with PBST and then treated them with a diluted solution of mouse or rabbit immunoglobulin G (IgG) secondary antibody at room temperature for one hour. Imaging was performed using the FluorChem E system from Cell Biosciences.

For immunofluorescence, we fixed the cells with 4% formaldehyde once the cell density met the experimental requirements, followed by blocking with a 5% BSA solution. The primary antibody dilution for the pre-stained target protein was prepared, and the cell coverslips were stored overnight at 4 ℃ in a humid dark box. The next day, fluorescent secondary antibody was applied to the slides to ensure complete coverage of the cells on the coverslips. Finally, the coverslips were mounted using a DAPI-containing mounting medium, and images were captured using a confocal microscope.

The primary antibodies used included Caspase 3 (CST #9662s) antibody, cleaved-caspase 3 (CST #9664s) antibody, STAT3 (CST #9139s) antibody, p-STAT3 (CST #9145s) antibody, PARP (CST #9532s) antibody, Cleaved-PARP (CST #5625s) antibody, CD133 (CST #64326s), Nestin (CST #33475s), Rac1 (CST #4651), MMP2 (CST #4022), EHMT2 (Abcam, ab18505, ab183889), NFκB (Abcam, ab305263). Bax (sc-7480) antibody and Bcl2 (sc-509) antibody were from Santa Cruz (Texas, CA, USA). GAPDH antibody was provided by Zhongshan Golden Bridge Biotechnology (Beijing, China).

Cell Counting Kit-8 (CCK-8) cell proliferation assay

The cells were prepared into single-cell suspension and seeded into a 96-well plate. They were cultured in an incubator with a concentration of 5% CO₂ at 37 ℃ until the cells attached on the wall. Removed the culture medium in the 96-well plate and prepared three culture mediums with different drug concentrations. Put them into the incubator for further culture for 12, 24, 48, and 72 h. The 10 µL CCK-8 reagent was added at these points and continued incubation for 1–4 hours, then the absorbance was measured at 450 nm with a microplate analyzer, and the data were analyzed by GraphPad.

Trans-well assay

The invasion of cells was evaluated by the transwell. The chamber was added to the 24-well plate and added 40 µL Matrigel into the upper chamber. After three hours, the chamber was removed, 600 µL high sugar medium containing 10% FBS was added to each well of 24-well plate. Then 100 µL of serum-free medium and 5×10⁴ cells were added into the upper chamber. The next day, cells were fixed with 4% paraformaldehyde for 15 min, dyed with 0.1% crystal violet for 20 minutes. Finally, cells were counted under the microscope.

Neurosphere formation assay

Glioma stem cells (GSCs) were treated with BIX-01294 at indicated concentrations (0, 1 µM for U87; 0, 5 µM for SHG140) and seeded at 1,000 cells per well in 96-well plates, with five replicates per condition. Neurosphere number and size were measured on day 7 following seeding.

In vitro limiting dilution assay

GSCs were plated in 96-well plates at varying densities (5, 10, 20, 50, 100, 200, or 500 cells per well) under different drug concentration gradients, with five replicates per condition. After 7 days, the number of tumorspheres in each well was recorded, and sphere formation efficiency was determined using Extreme Limiting Dilution Analysis (ELDA; http://bioinf.wehi.edu.au/software/elda).

Co-immunoprecipitation (Co-IP)

Total cell lysates were obtained on ice using RIPA lysis buffer supplemented with protease inhibitors. A small aliquot was retained as input and centrifuged at 12,000 ×g for 10 minutes. The remaining lysate was incubated overnight at 4 ℃ with the EHMT2 primary antibody (Abcam, ab183889), with mouse or rabbit IgG used as negative controls. The next day, magnetic beads were added to capture the immune complexes for 2 hours at 4 ℃. After washing with PBS, the bead-bound NFκB (Abcam, ab16502) was examined by western blot analysis.

Statistical analysis

The bar chart represents the mean standard deviation from at least three experimental replicates. The number of repeated experiments involved is n=3. Most of the experiments were statistically analyzed using Student’s t-test. The data were analyzed by GraphPad prism 6. Significance of p values were set at ^NSP>0.05, *P<0.05, **P<0.01, ***P<0.001. ****P<0.0001.

Results

Expression of EHMT2 is associated with GBM and GBM subtype

EHMT2 is a histone-modifying enzyme that is highly abundant in the nucleus. We presented the expression distribution of EHMT2 in different tumors based on TCGA data (Figure 1A); based on CGGA data, we demonstrated the expression of EHMT2 in different subtypes (Figure 1B); By analyzing the molecular subtypes of GBM in TCGA and CGGA, we found that the expression of EHMT2 was relatively high in the classical subtype and pre neuronal subtype, indicating a close relationship between EHMT2 and the growth and development of GBM (Figure 1C). We found that EHMT2 was expressed in U87 and the primary cell line SHG140 and was concentrated in the nucleus. It was also expressed in stem cells of U87 and SHG140 (Figure 1D,1E).

Figure 1 Expression of EHMT2 in GBM and its molecular subtypes. (A) Expression of EHMT2 in various tumors. (B) Expression of EHMT2 in different glioma subtypes based on CGGA data. (C) The expression features in GBM subtypes were also explored with the TCGA and CGGA datasets. (D,E) EHMT2 expression in U87, SHG140 and their stem cells detected by immunofluorescence. Scale bar: 20µm in (D); 50 µm in (E). AC, adenocarcinoma; CGGA, Chinese Glioma Genome Atlas; EHMT2, Euchromatic Histone Lysine Methyltransferase 2; pGBM, primary glioblastoma; pTPM, primary transcripts per million; TCGA, The Cancer Genome Atlas.

BIX-01294 inhibited the proliferation and migration of GBM cells

To evaluate the effect of EHMT2 on GBM cells, BIX-01294 was selected as an inhibitor of EHMT2. We first studied the effect of BIX-01294 on the survival rate of GBM cell lines and found that the semi maximal inhibitory concentration (IC₅₀) of SHG140 was approximately 5 µM, while the semi maximal inhibitory concentration of U87 was 1 µM (Figure 2A). Based on this discovery, we selected two concentrations of SHG140 (2.5 and 5 µM) and U87 (0.5 and 1 µM) to treat GBM cells, and measured cell survival rates at 24 and 48 hours; the results showed that BIX-01294 inhibited the proliferation of GBM cells (Figure 2B). In addition, we found that BIX-01294 can inhibit the migration of GBM cells. Based on the survival rate of GBM cells exceeding 90% after low concentration administration (U87 0.5 µM, SHG140 2.5 µM) (Figure 2C), the difference between the values of the treatment group and the control group was statistically significant (Figure 2D).

Figure 2 BIX-01294 inhibited the proliferation and migration of GBM cells. (A) U87 and SHG140 were treated with BIX-01294 for 24 h, and cell viability was detected using CCK-8 assay. (B) Cells treated with BIX-01294, 0.5 μM, 1 μM in U87 and 2.5 μM, 5 μM in SHG140 for 24 and 48 h. Data are shown as the mean ± 95% CI, n=3, Bonferroni correction, ****P<0.0001, Two-way ANOVA. (C) Transwell assay was used to evaluate the effect of BIX-01294 on GBM cell migration (staining method: crystal violet staining). (D) Perform statistical analysis on the data of (C). Data are shown as the mean ± SD. n=3, *P<0.05, **P<0.01, Student’s t-test. ANOVA, analysis of variance; CCK-8, Cell Counting Kit-8; CI, confidence interval; GBM, glioblastoma; SD, standard deviation.

Since targeting EHMT2 inhibited GBM cell proliferation and migration, we analyzed the expression of apoptosis and migration-related proteins in each treatment group. We found that following BIX-01294 treatment, the expression of cleaved-caspase 3, cleaved-PARP, and Bax increased, and that of Bcl2 decreased in a concentration-dependent manner, suggesting that the inhibition of EHMT2 could promote GBM apoptosis; the expression of MMP2 and Rac1 showed that targeting EHMT2 suppressed GBM cell migration (Figure 3A). We also evaluated the Pearson correlation between EHMT2 and invasion and proliferation related genes. The data showed that EHMT2 had a stronger correlation with these genes (Figure 3B,3C).

Figure 3 The relationship between EHMT2 and the expression of invasion and proliferation related proteins in GBM. (A) Cells treated with BIX-01294 in different doses. Western blot was used to detect cell apoptosis and migration-related protein expressions. (B) Pearson correlation analysis between EHMT2 and cell apoptosis and migration-related gene expressions in TCGA data sets. (C) The supplementation of section B using CGGA data distinguishes glioma of different grades. CGGA, Chinese Glioma Genome Atlas; GBM, glioblastoma; EHMT2, Euchromatic Histone Lysine Methyltransferase 2; TCGA, The Cancer Genome Atlas; WHO, World Health Organization.

EHMT2 inhibits self-renewal of GSCs

As mentioned earlier, EHMT2 is highly expressed in the pre neuronal subtype, making it particularly crucial to detect the role of EHMT2 in GSCs. Single cell spheroidization experiments showed that after treatment with BIX-01294, the spheroidization and self-renewal abilities of GSCs decreased, and the differences between different concentration groups were significant (Figure 4A-4C). CCK-8 also confirmed the inhibitory effect of BIX-01294 on GSC (Figure 4D). Cell ball staining for stem cell markers CD133 and Nestin also indicated that inhibition of EHMT2 can reduce the self-renewal of GSC (Figure 4E).

Figure 4 BIX-01294 inhibited self-renewal of U87s and SHG140s. (A) Single cell spheroidization assay for detecting drug effects. (B) Extreme dilution experiment to detect the effect of drugs on the self-renewal ability of GSC. (C,D) Statistical analysis of differences in (A) and (B) results. Data is shown as the mean ± 95% CI, n=5, *P<0.05, ****P<0.0001, Student’s t-test. (E) Immunofluorescence detection of the expression of dry markers in cell spheroids under drug action. Scale bar: 20 µm. CI, confidence interval; GSC, glioma stem cell.

Association of EHMT2 with cancer-related pathways including STAT3 pathway

We explored whether EHMT2 is involved in the regulation of some classical pathways. We found that EHMT2 reduced the phosphorylation level of STAT3 (Figure 5A). PPI protein interaction network analysis suggests that EHMT2 may regulate the STAT3 signaling pathway through NFκB1 (Figure 5B). In addition, based on the data from TCGA and Gene Expression Omnibus (GEO) databases, we studied the correlation between EHMT2 and STAT3, NOTCH1, NFκB1 pathways by Pearson correlation analysis and found that EHMT2 is highly correlated with these pathways (Figure 5C,5D). Further experimental results suggest that EHMT2 may interact with NFκB1 (Figure 5E,5F). Therefore, we hypothesize that EHMT2 may activate the STAT3 signaling pathway by interacting with NFκB. Further studies will be needed to experimentally validate this hypothesis.

Figure 5 EHMT2 is associated with STAT3 and other classical pathway makers. (A) Cells treated with BIX-01294 in different doses. STAT3 and phosphorylation of STAT3 expression were detected by Western blot. (B) PPI protein interaction network analysis suggests the distribution of proteins that may interact with EHMT2. (C,D) Pearson correlation analysis between EHMT2 and STAT3, NOTCH1, NFκB1 in TCGA data sets and GSE23806. (E) The colocalization of EHMT2 and NFκB in SHG140 cells was detected by immunofluorescence staining. Scale bar: 10 µm. (F) The binding relationship between EHMT2 and NFκB was detected by co-immunoprecipitation. EHMT2, Euchromatic Histone Lysine Methyltransferase 2; IgG, immunoglobulin G; IP, immunoprecipitation; NFκB, nuclear factor kappa-B; PPI, protein-protein interaction; STAT3, signal transducer and activator of transcription 3; TCGA, The Cancer Genome Atlas.

Similarly, we analyzed and screened the gene sets showing a high correlation with EHMT2 in TCGA and CGGA databases. Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we found that the genes that were highly correlated with EHMT2 were involved in apoptosis, invasion, and migration of GBM. EHMT2 high correlated genes were associated with the positive regulation of cell adhesion, extracellular matrix organization and cell-substrate adhesion, suggesting a strong correlation between EHMT2 and GBM invasion and migration. In addition, the regulation of tumor necrosis factor superfamily cytokine production and the negative regulation of cell death by EHMT2 suggest that EHMT2 is closely related to GBM apoptosis (Figure 6). These results also confirm our previous experimental results.

Figure 6 Bioinformatics analysis of the biological characteristics and signaling pathways highly correlated with EHMT2. (A,B) KEGG pathway analysis of EHMT2 related genes in TCGA and CGGA data sets. The P value threshold and FDR correction are indicated in the figure. BP, biological process; CGGA, Chinese Glioma Genome Atlas; DE gene, differentially expressed gene; EHMT2, Euchromatic Histone Lysine Methyltransferase 2; FDR, false discovery rate; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; Sig, significant; TCGA, The Cancer Genome Atlas.

Discussion

EHMT2 is mainly responsible for the methylation of H3K9 and H3K27 proteins in the euchromatin region (9). H3K9 methylation in the euchromatin region was found to be significantly reduced after the knockdown of the EHMT2 gene (10). Recent studies have found that the imbalance of H3K9 methylation caused by the abnormal expression of EHMT2 plays a vital role in the occurrence of tumors (11). This study found that EHMT2 is highly expressed in GBM cells and plays a crucial role in promoting cancer invasion and metastasis. Abnormal expressions of G9a can directly lead to gene mutation, amplification, or the expression and inactivation of some critical proteins, eventually leading to tumors. Through the KEGG analysis of TCGA and CGGA databases, EHMT2 was confirmed to be related to tumor invasion and apoptosis. We further found that EHMT2 is associated with multiple GBM-related signaling pathways, including the STAT3 pathway. These findings reveal a novel approach to prevent GBM recurrence by targeting the EHMT2/STAT3 axis.

Since the high-throughput screening of 125,000 compounds conducted by Kubicek et al. in 2007, BIX-01294 as an EHMT2 specific inhib©itor, has been one of the hot topics in the study of tumors (6), because it can restrain the specificity of EHMT2 activity, adjust histone H3K9 methylation, change the spatial structure of chromatin, and induce of tumor suppressor gene expression, making the growth of malignant tumor cells more manageable (12). Unfortunately, although some studies have verified the effect of BIX-01294 in subcutaneous GBM transplantation (13), no literature has been found in the intracranial Orthotopic Implanted Tumor Model. In summary, BIX-01294 regulates tumor proliferation, invasion, and apoptosis. In terms of glioma, it has been shown in the literature that glioma cells treated with BIX-01294 show increased sensitivity to temozolomide, suggesting the great potential of this drug in clinical settings (14).

The clinical treatment of glioma is challenging, making it necessary to conduct further research for the development of novel therapeutic strategies. There are relatively few studies in literature on EHMT2 and its specific inhibitor, BIX-01294. At the beginning of this study, EHMT2 was found to be highly expressed in GBM. Therefore, we suspected whether BIX-01294 has an inhibitory effect on GBM. Our study showed that after treatment with BIX-01294, GBM cell proliferation and invasion ability decreased, the apoptosis level increased. There were significant differences in the changes of invasion-related proteins and apoptosis-related proteins. These results indicated that BIX-01294 inhibited the growth of GBM cells by inhibiting EHMT2. The inhibitory effect was significantly increased with an increase in drug concentration. Some valuable previous studies also support our findings, by demonstrating: BIX-01294 to possess anticancer activity in breast cancer, colon cancer, leukemia, human germ cell tumors and cervical squamous carcinoma, and the antitumor effect of BIX-01294 was related to inhibition of the histone methyltransferase effect of EHMT2 (15).

Similarly, we detected alterations in the expression of STAT3 and phosphorylated-STAT3 (p-STAT3) in the cells. STAT3 plays an important role in transmitting the signals of cytokines and growth factors, especially in a variety of human malignant tumors, including glioma (16-18). Our results indicate that the level of STAT3 does not show significant changes, but the level of p-STAT3 is significantly reduced. We also found that EHMT2 interacts with NFκB, which may be crucial for the regulation of STAT3 activity by EHMT2. Additionally, we observed that EHMT2 is associated with multiple tumor-related signaling pathways. Similar results suggest that targeted inhibition of a variety of signaling pathways including STAT3, Myc, NFκB, Wnt, ERK1 and mTOR can achieve the purpose of antitumor effects (19-21). In addition, EHMT2 is strongly correlated with biological processes such as intercellular adhesion and tumor necrosis factor production. These findings indicate that EHMT2 may be a critical factor in tumor-targeted therapy. Regarding the limitations of this study: we have not yet conducted experiments on the targeted inhibitory effect of BIX-01294 in the orthotopic xenograft mouse model, nor have we explored how BIX-01294 crosses the blood-brain barrier or its in vivo toxicity. These aspects need to be addressed in future experiments.

Conclusions

In this study, we discovered the role of BIX-01294 as an EHMT2 inhibitor in GBM cells, and its potential for clinical treatment, providing an experimental foundation for the development of novel clinical interventions for GBM. However, more research on EHMT2 and BIX-01294 is needed, such as that exploring the effect of BIX-01294 on the autophagy of GBM cells, the adverse reactions and side effects of BIX-01294 therapy, the potential for the development of drug resistance by cancer cells, the possibility of combination with temozolomide, and the specific role of EHMT2 in GBM cells.

Acknowledgments

The authors would like to thank the researchers who provided open access to the raw data.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0235/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0235/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0235/prf

Funding: The study was supported by the Health and Family Planning Research Project of Pudong New Area Health Commission (Project No. PW2022A - 27, to L.Z.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2026-1-0235/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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