Study on Connexin43-Mediated Temozolomide Resistance in Glioma through Activation of the E2F1/ERCC1 Axis

Academic Background

Glioma is the most common and fatal tumor of the central nervous system, with temozolomide (TMZ) being the standard treatment. However, TMZ therapy often leads to tumor recurrence and drug resistance, severely limiting its efficacy. Tumor-associated astrocytes (TAAs) are a key component of the tumor microenvironment, and increasing evidence suggests that abnormal expression of Connexin43 (Cx43) is closely related to glioma progression and TMZ resistance. However, the specific mechanisms by which Cx43 mediates TMZ resistance through interactions between glioma and astrocytes remain unclear. Therefore, this study aims to explore how Cx43 regulates DNA repair mechanisms to induce TMZ resistance in the context of glioma-astrocyte interactions.

Source of the Paper

This study was conducted by Yanping Gui, Hongkun Qin, Xinyu Zhang, and others, primarily from the School of Basic Medicine and Clinical Pharmacy at China Pharmaceutical University, with some authors affiliated with the Department of Pathology at Nanjing Drum Tower Hospital, Medical School of Nanjing University. The research was published online on November 8, 2024, in the journal Neuro-Oncology, under the title “Glioma–astrocyte connexin43 confers temozolomide resistance through activation of the E2F1/ERCC1 axis.”

Research Process and Results

1. Expression of Cx43 in Glioma and Its Relationship with TMZ Resistance

The study first confirmed through immunohistochemistry (IHC) and Western blotting that Cx43 expression is significantly higher in glioma tissues compared to normal brain tissues and increases with the pathological grade of glioma. Analysis of the Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) databases revealed that the Cx43-encoding gene GJA1 is most highly expressed in recurrent glioma patients, and its high expression is significantly associated with poor prognosis. These results indicate that high Cx43 expression is closely related to TMZ resistance in glioma.

2. Astrocytes Mediate TMZ Resistance in Glioma through Cx43

To investigate how astrocytes mediate TMZ resistance in glioma through Cx43, the study established a co-culture model of glioma cells and astrocytes. The results showed that astrocytes significantly upregulate Cx43 expression in glioma cells through direct contact, enhancing their resistance to TMZ. Further treatment of co-cultured cells with Cx43 inhibitors Carbenoxolone (CBX) and Meclofenamate (Meclo) revealed that these inhibitors could reverse the protective effects of astrocytes, significantly increasing TMZ cytotoxicity.

3. Cx43 Regulates TMZ Resistance through the β-catenin/miR-205-5p/E2F1 Axis

To delve deeper into the molecular mechanisms by which Cx43 mediates TMZ resistance, the study used co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify a direct interaction between Cx43 and β-catenin. Further experiments demonstrated that Cx43 binds to β-catenin through its C-terminal domain, sequestering β-catenin at the cell membrane and inhibiting its nuclear translocation, thereby reducing the transcriptional activity of T-cell factor/lymphoid enhancer factor (TCF/LEF). This suppression leads to downregulation of miR-205-5p expression, subsequently activating its target gene E2F1.

4. E2F1 Mediates TMZ Resistance by Upregulating ERCC1

E2F1 is an important transcription factor, and the study confirmed through bioinformatics analysis and chromatin immunoprecipitation (ChIP) experiments that E2F1 directly binds to the promoter region of the ERCC1 gene, promoting its transcription. ERCC1 is a key protein in the nucleotide excision repair (NER) pathway, and its high expression enhances TMZ resistance in glioma cells. Both in vitro and in vivo experiments confirmed that E2F1 mediates TMZ resistance in glioma cells by upregulating ERCC1 expression.

5. Role of the Cx43/miR-205-5p/E2F1/ERCC1 Axis in TMZ Resistance

Finally, the study found that knocking down Cx43 expression significantly reduced E2F1 and ERCC1 expression, markedly increasing glioma cell sensitivity to TMZ. Further in vivo experiments showed that Cx43 knockout mice exhibited significantly prolonged survival and inhibited tumor growth under TMZ treatment. These results confirm that Cx43 mediates TMZ resistance in glioma through the miR-205-5p/E2F1/ERCC1 axis.

Research Conclusions and Significance

This study reveals a novel mechanism by which astrocytes in the glioma microenvironment upregulate Cx43 expression, activating the E2F1/ERCC1 axis to enhance DNA repair capacity, leading to TMZ resistance. This discovery provides a new therapeutic target for glioma, suggesting that inhibiting Cx43 or its downstream signaling pathways may effectively improve TMZ efficacy. Additionally, the study proposes the Cx43/miR-205-5p/E2F1/ERCC1 axis as a key regulatory network in TMZ resistance, offering a theoretical foundation for developing new treatment strategies.

Research Highlights

1. Novel Molecular Mechanism: This study is the first to reveal that Cx43 mediates TMZ resistance in glioma through the β-catenin/miR-205-5p/E2F1/ERCC1 axis, filling a gap in this field of research.
2. Multi-level Experimental Validation: The study validated the role of Cx43 in TMZ resistance through in vitro cell experiments, in vivo animal models, and clinical sample analysis, ensuring highly reliable results.
3. Potential Therapeutic Target: Cx43 and its downstream signaling pathways may serve as new therapeutic targets, offering new strategies to overcome TMZ resistance.
4. Clinical Significance: The findings suggest that high Cx43 expression could serve as a biomarker for predicting TMZ treatment outcomes in glioma patients, aiding in the development of personalized treatment plans.

Other Valuable Information

The study also found that Cx43 expression is closely related to the invasiveness and malignancy of glioma, suggesting that Cx43 may play a similar regulatory role in other types of tumors. Additionally, the novel Cx43 inhibitors CBX and Meclo used in the study showed promising effects in reversing TMZ resistance, potentially paving the way for future clinical drug development.

Summary

This study thoroughly explores the molecular mechanisms by which astrocytes in the glioma microenvironment mediate TMZ resistance through Cx43, highlighting the critical role of the Cx43/miR-205-5p/E2F1/ERCC1 axis in glioma treatment. The research not only provides new therapeutic targets for glioma but also lays the theoretical groundwork for developing strategies to overcome TMZ resistance.