Abstract
Immune evasion is a major barrier to effective glioma treatment, yet the molecular drivers contributing to this process remain insufficiently understood. Engrailed-1 (EN1), a developmental transcription factor, has recently emerged as a potential oncogenic regulator, but its role in glioma immune modulation is unclear. This study aimed to comprehensively investigate the function of EN1 in glioma, with a particular focus on its involvement in immune evasion, by integrating bulk and single-cell transcriptomic analyses together with ceRNA regulatory network construction and experimental validation. EN1 expression, diagnostic value, and prognostic significance were assessed using TCGA, GTEx, and CGGA datasets. Immune infiltration and microenvironmental features were evaluated through CIBERSORT, ESTIMATE, and WGCNA. Single-cell RNA-seq data (GSE182109) were used to characterize the cell-type distribution and developmental trajectory of EN1. A NEAT1/miR-9-5p/miR-128-3p/EN1 ceRNA axis was constructed using multi-database predictions. Functional assays including Western blot, CCK-8, and Transwell experiments were performed to validate EN1’s effects in glioblastoma cells. EN1 was markedly overexpressed in glioma and associated with poorer survival. Elevated EN1 expression correlated with increased infiltration of immunosuppressive cells, reduced tumor purity, and higher immune checkpoint expression. Single-cell analysis revealed progressive EN1 upregulation along malignant cell pseudotime. Drug sensitivity prediction suggested that the EN1-high group may have reduced sensitivity to temozolomide and additional chemotherapeutic agents. The identified NEAT1/miR-9-5p/miR-128-3p/EN1 ceRNA loop suggested a regulatory mechanism contributing to EN1 activation. EN1 knockdown significantly suppressed glioblastoma cell proliferation and invasion in vitro. EN1 is associated with glioma aggressiveness, immune microenvironmental features, and predicted chemotherapeutic response, and a putative NEAT1/miR-9-5p/miR-128-3p/EN1 axis may contribute to EN1 dysregulation. These findings identify EN1 as a promising biomarker and potential therapeutic target for improving glioma treatment.
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Data availability
The bulk RNA-seq and corresponding clinical data analyzed in this study were obtained from The Cancer Genome Atlas (TCGA, https://portal.gdc.cancer.gov/), including the TCGA-GBM and TCGA-LGG cohorts. Normal brain tissue expression data were retrieved from the Genotype-Tissue Expression (GTEx) project (https://gtexportal.org). An independent validation cohort was obtained from the Chinese Glioma Genome Atlas (CGGA, http://www.cgga.org.cn/), specifically the CGGA_693 dataset. Single-cell RNA sequencing data were downloaded from the Gene Expression Omnibus (GEO) database under accession number GSE182109. All datasets used in this study are publicly available and fully de-identified.
References
Ostrom, Q. T. et al. CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the united States in 2016–2020. Neuro Oncol. 25, iv1–iv99. https://doi.org/10.1093/neuonc/noad149 (2023).
Louis, D. N. et al. The 2021 WHO classification of tumors of the central nervous system: a summary. Neuro Oncol. 23, 1231–1251. https://doi.org/10.1093/neuonc/noab106 (2021).
Grochans, S. et al. Epidemiology of glioblastoma Multiforme-Literature review. Cancers (Basel). 14 https://doi.org/10.3390/cancers14102412 (2022).
Kotecha, R., Odia, Y., Khosla, A. A. & Ahluwalia, M. S. Key clinical principles in the management of glioblastoma. JCO Oncol. Pract. 19, 180–189. https://doi.org/10.1200/OP.22.00476 (2023).
Ntafoulis, I., Koolen, S. L. W., Leenstra, S. & Lamfers, M. L. M. Drug Repurposing, a Fast-Track approach to develop effective treatments for glioblastoma. Cancers (Basel). 14. https://doi.org/10.3390/cancers14153705 (2022).
Thenuwara, G., Curtin, J. & Tian, F. Advances in diagnostic tools and therapeutic approaches for gliomas: A comprehensive review. Sens. (Basel). 23 https://doi.org/10.3390/s23249842 (2023).
Miller, K. D. et al. Cancer treatment and survivorship statistics, 2022. CA Cancer J. Clin. 72, 409–436. https://doi.org/10.3322/caac.21731 (2022).
Sharma, P., Aaroe, A., Liang, J. & Puduvalli, V. K. Tumor microenvironment in glioblastoma: current and emerging concepts. Neurooncol Adv. 5, vdad009. https://doi.org/10.1093/noajnl/vdad009 (2023).
Zhang, X. et al. The immunosuppressive microenvironment and immunotherapy in human glioblastoma. Front. Immunol. 13, 1003651. https://doi.org/10.3389/fimmu.2022.1003651 (2022).
Sgado, P. et al. Slow progressive degeneration of nigral dopaminergic neurons in postnatal engrailed mutant mice. Proc. Natl. Acad. Sci. U S A. 103, 15242–15247. https://doi.org/10.1073/pnas.0602116103 (2006).
Simon, H. H., Saueressig, H., Wurst, W. & Goulding, M. D. O’Leary, D. D. Fate of midbrain dopaminergic neurons controlled by the engrailed genes. J. Neurosci. 21, 3126–3134. https://doi.org/10.1523/JNEUROSCI.21-09-03126.2001 (2001).
Xu, J. et al. Engrailed-1 promotes pancreatic cancer metastasis. Adv. Sci. (Weinh). 11, e2308537. https://doi.org/10.1002/advs.202308537 (2024).
Cui, Y. et al. EN1 promotes lung metastasis of salivary adenoid cystic carcinoma by regulating the PI3K-AKT pathway and epithelial-mesenchymal transition. Cancer Cell. Int. 24, 51. https://doi.org/10.1186/s12935-024-03230-7 (2024).
Peluffo, G. et al. EN1 is a transcriptional dependency in Triple-Negative breast cancer associated with brain metastasis. Cancer Res. 79, 4173–4183. https://doi.org/10.1158/0008-5472.CAN-18-3264 (2019).
Zhao, N. et al. [En1 promotes cell proliferation and migration via Hedgehog signaling pathway in esophageal squamous cell carcinoma]. Zhonghua Zhong Liu Za Zhi. 46, 99–107. https://doi.org/10.3760/cma.j.cn112152-20231026-00257 (2024).
Kanehisa, M. & Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30. https://doi.org/10.1093/nar/28.1.27 (2000).
Zagozewski, J. L., Zhang, Q., Pinto, V. I., Wigle, J. T. & Eisenstat, D. D. The role of homeobox genes in retinal development and disease. Dev. Biol. 393, 195–208. https://doi.org/10.1016/j.ydbio.2014.07.004 (2014).
Xu, F. et al. HOXD13 suppresses prostate cancer metastasis and BMP4-induced epithelial-mesenchymal transition by inhibiting SMAD1. Int. J. Cancer. 148, 3060–3070. https://doi.org/10.1002/ijc.33494 (2021).
Gao, A. C., Lou, W. & Isaacs, J. T. Down-regulation of homeobox gene GBX2 expression inhibits human prostate cancer clonogenic ability and tumorigenicity. Cancer Res. 58, 1391–1394 (1998).
Lin, L. et al. The homeobox transcription factor MSX2 partially mediates the effects of bone morphogenetic protein 4 (BMP4) on somatic cell reprogramming. J. Biol. Chem. 293, 14905–14915. https://doi.org/10.1074/jbc.RA118.003913 (2018).
Mantovani, A., Sozzani, S., Locati, M., Allavena, P. & Sica, A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 23, 549–555. https://doi.org/10.1016/s1471-4906(02)02302-5 (2002).
Yang, Y. et al. M2 Macrophage-Derived exosomes promote angiogenesis and growth of pancreatic ductal adenocarcinoma by targeting E2F2. Mol. Ther. 29, 1226–1238. https://doi.org/10.1016/j.ymthe.2020.11.024 (2021).
Chen, Y., Zhang, S., Wang, Q. & Zhang, X. Tumor-recruited M2 macrophages promote gastric and breast cancer metastasis via M2 macrophage-secreted CHI3L1 protein. J. Hematol. Oncol. 10, 36. https://doi.org/10.1186/s13045-017-0408-0 (2017).
Tan, S. et al. Transcription factor Zhx2 is a checkpoint that programs macrophage polarization and antitumor response. Cell. Death Differ. 30, 2104–2119. https://doi.org/10.1038/s41418-023-01202-4 (2023).
Jiang, H. et al. Zeb1-induced metabolic reprogramming of Glycolysis is essential for macrophage polarization in breast cancer. Cell. Death Dis. 13, 206. https://doi.org/10.1038/s41419-022-04632-z (2022).
Brunner-Weinzierl, M. C. & Rudd, C. E. CTLA-4 and PD-1 control of T-Cell motility and migration: implications for tumor immunotherapy. Front. Immunol. 9, 2737. https://doi.org/10.3389/fimmu.2018.02737 (2018).
Sun, C., Mezzadra, R. & Schumacher, T. N. Regulation and function of the PD-L1 checkpoint. Immunity 48, 434–452. https://doi.org/10.1016/j.immuni.2018.03.014 (2018).
Rowshanravan, B., Halliday, N. & Sansom, D. M. CTLA-4: a moving target in immunotherapy. Blood 131, 58–67. https://doi.org/10.1182/blood-2017-06-741033 (2018).
Chen, X. et al. Oct4A palmitoylation modulates tumorigenicity and stemness in human glioblastoma cells. Neuro Oncol. 25, 82–96. https://doi.org/10.1093/neuonc/noac157 (2023).
Sun, Y. M. et al. Enhancer-driven transcription of MCM8 by E2F4 promotes ATR pathway activation and glioma stem cell characteristics. Hereditas 160, 29. https://doi.org/10.1186/s41065-023-00292-x (2023).
Bowen, C., Zheng, T. & Gelmann, E. P. NKX3.1 suppresses TMPRSS2-ERG gene rearrangement and mediates repair of androgen Receptor-Induced DNA damage. Cancer Res. 75, 2686–2698. https://doi.org/10.1158/0008-5472.CAN-14-3387 (2015).
Soret, C. et al. Distinct mechanisms for opposite functions of homeoproteins Cdx2 and HoxB7 in double-strand break DNA repair in colon cancer cells. Cancer Lett. 374, 208–215. https://doi.org/10.1016/j.canlet.2016.02.026 (2016).
Liu, X. A. B. C. & Family Transporters Adv. Exp. Med. Biol. 1141, 13–100, doi:https://doi.org/10.1007/978-981-13-7647-4_2 (2019).
Vadlapatla, R. K., Vadlapudi, A. D., Pal, D. & Mitra, A. K. Mechanisms of drug resistance in cancer chemotherapy: coordinated role and regulation of efflux transporters and metabolizing enzymes. Curr. Pharm. Des. 19, 7126–7140. https://doi.org/10.2174/13816128113199990493 (2013).
Xu, J., Xu, J., Liu, X. & Jiang, J. The role of lncRNA-mediated CeRNA regulatory networks in pancreatic cancer. Cell. Death Discov. 8, 287. https://doi.org/10.1038/s41420-022-01061-x (2022).
Tay, Y., Rinn, J. & Pandolfi, P. P. The multilayered complexity of CeRNA crosstalk and competition. Nature 505, 344–352. https://doi.org/10.1038/nature12986 (2014).
Zhang, Y., Luo, M., Cui, X., O’Connell, D. & Yang, Y. Long noncoding RNA NEAT1 promotes ferroptosis by modulating the miR-362-3p/MIOX axis as a CeRNA. Cell. Death Differ. 29, 1850–1863. https://doi.org/10.1038/s41418-022-00970-9 (2022).
Wen, S. et al. Long non-coding RNA NEAT1 promotes bone metastasis of prostate cancer through N6-methyladenosine. Mol. Cancer. 19, 171. https://doi.org/10.1186/s12943-020-01293-4 (2020).
Wang, J. et al. N6-methyladenosine reader hnRNPA2B1 recognizes and stabilizes NEAT1 to confer chemoresistance in gastric cancer. Cancer Commun. (Lond). 44, 469–490. https://doi.org/10.1002/cac2.12534 (2024).
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We are grateful to all the members of our study.
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This work was supported by grants from High-Level Talent Scientific Research Start-Up Project of the Affiliated Hospital of Guangdong Medical University [grant number GCC2021006].
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ZL and WL: Conceptualization, Writing - Review & Editing. ZJ: Methodology, Writing- Original draft. YW and JY: Formal analysis. LZ and XL: Visualization. TW: Data Curation. All the authors read and approved the final manuscript.
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Jia, Z., Wang, Y., Yao, J. et al. Engrailed 1 promotes immune evasion and chemoresistance in glioma through single cell and CeRNA network analyses. Sci Rep (2026). https://doi.org/10.1038/s41598-026-35553-y
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DOI: https://doi.org/10.1038/s41598-026-35553-y
