OY-TES-1 Splice Variant V5a in Glioma: A Driver of Malignancy and Potential Therapeutic Target

Abstract

Objective

Glioma is a highly lethal tumor of the central nervous system (CNS) with limited therapeutic options. Recent evidence has highlighted the role of dysregulated alternative splicing in glioma progression. Although OY-TES-1 has been proposed as a potential therapeutic target, its splice isoforms have not been fully characterized. This study aimed to identify the clinically relevant splice variant of OY-TES-1 associated with glioma progression and to evaluate its potential as a target for innovative therapeutic strategies against this challenging disease.

Method

The potential splicing patterns of OY-TES-1, along with their relative frequency and correlation with patient survival, were analyzed via the TCGA SpliceSeq and OncoSplicing databases. RNA-Seq by expectation maximization (RSEM) values and clinicopathological data for all OY-TES-1 gene transcripts were downloaded from the UCSC Xena database, and Cox regression analysis was performed for both univariate and multivariate prognostic assessments. The expression of OY-TES-1 mRNA in glioma and normal brain tissues was detected via RT-PCR. The relationships between OY-TES-1 mRNA expression and the clinicopathological characteristics of glioma patients were analyzed via the χ² test. OY-TES-1-V5a was overexpressed in glioma cells through transient transfection with plasmids as well as stable transfection with lentivirus for further functional analysis. Glioma cell proliferation was assessed via the Cell Counting Kit-8 (CCK-8) assay. Migration and invasion abilities were evaluated via wound healing, Transwell, and Transwell Matrigel assays. Apoptosis was analyzed by flow cytometry.

Results

Bioinformatic analysis revealed four alternative splice variants of OY-TES-1 in glioma, among which OY-TES-1-V5a presented a relatively high percent spliced-in (PSI) value that was associated with significantly shorter overall survival. OY-TES-1-V5a was further identified as an independent prognostic risk factor for glioma patients, as its mRNA expression was significantly associated with Karnofsky performance status (KPS), tumor grade, and isocitrate dehydrogenase 1 (IDH1) mutation status. RT-PCR validation confirmed that OY-TES-1-V5a was overexpressed in glioma tissues compared with normal brain tissues. Functionally, forced expression of OY-TES-1-V5a enhanced glioma cell proliferation, migration, and invasion while suppressing apoptosis.

Conclusions

The OY-TES-1 splice variant V5a is highly expressed in glioma, is associated with poor prognosis, and actively drives malignant behavior, indicating its potential utility as a prognostic biomarker and a candidate target for therapeutic intervention.

Keywords

Glioma / OY-TES-1 / Alternative splicing / OY-TES-1-V5a / RNA splicing / Prognostic biomarker / Cancer antigen / Therapeutic target

References

[1] Ostrom QT, Price M, Neff C, et al. . CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the United States in 2015–2019. Neuro Oncol., 2022, 24(Suppl 5): v1-v95.

[2] Gautam M, Gabrani R. Current combinatorial therapeutic aspects: the future prospect for glioblastoma treatment. Curr Med Sci., 2024, 44(6): 1175-1184.

[3] Salvalaggio A, Pini L, Bertoldo A, et al. . Glioblastoma and brain connectivity: the need for a paradigm shift. Lancet Neurol., 2024, 23(7): 740-748.

[4] Li RC, Liu C, Wang GJ, et al. . ELMOD2 overexpression predicts adverse outcomes and regulates tumor progression in gliomas. Curr Med Sci., 2025, 45(3): 549-561.

[5] Ono T, Kurashige T, Harada N, et al. . Identification of proacrosin binding protein sp32 precursor as a human cancer/testis antigen. Proc Natl Acad Sci U S A., 2001, 98(6): 3282-3287.

[6] Whitehurst AW. Cause and consequence of cancer/testis antigen activation in cancer. Annu Rev Pharmacol Toxicol., 2014, 54: 251-272.

[7] Salmaninejad A, Zamani MR, Pourvahedi M, et al. . Cancer/testis antigens: expression, regulation, tumor invasion, and use in immunotherapy of cancers. Immunol Invest., 2016, 45(7): 619-640.

[8] Ren S, Zhang Z, Li M, et al. . Cancer testis antigen subfamilies: attractive targets for therapeutic vaccine (Review). Int J Oncol., 2023, 62(6): 71

[9] Bradley RK, Anczuków O. RNA splicing dysregulation and the hallmarks of cancer. Nat Rev Cancer., 2023, 23(3): 135-155.

[10] Marasco LE, Kornblihtt AR. The physiology of alternative splicing. Nat Rev Mol Cell Biol., 2023, 24(4): 242-254.

[11] Wang E, Aifantis I. RNA splicing and cancer. Trends Cancer., 2020, 6(8): 631-644.

[12] Seiler M, Yoshimi A, Darman R, et al. . H3B–8800, an orally available small-molecule splicing modulator, induces lethality in spliceosome-mutant cancers. Nat Med., 2018, 24(4): 497-504.

[13] Zhang Y, Qian J, Gu C, et al. . Alternative splicing and cancer: a systematic review. Signal Transduct Target Ther., 2021, 6(1): 78

[14] Luo B, Yun X, Li J, et al. . Cancer-testis antigen OY-TES-1 expression and immunogenicity in hepatocellular carcinoma. Curr Med Sci., 2020, 40(4): 719-728.

[15] Li X, Yan J, Fan R, et al. . Serum immunoreactivity of cancer/testis antigen OY-TES-1 and its tissues expression in glioma. Oncol Lett., 2017, 13(5): 3080-3086.

[16] Zeng X, Nong WX, Zou XQ, et al. . Prediction and identification of HLA-A*0201-restricted epitopes from cancer testis antigen CT23. Hum Vaccin Immunother., 2023, 19(3): 2293299

[17] Lin L, Zou X, Nong W, et al. . The potential value of cancer-testis antigens in ovarian cancer: Prognostic markers and targets for immunotherapy. Immun Inflamm Dis., 2024, 12(6e1284

[18] Ge YY, Zhang QM, Liu C, et al. . Combined treatment with epigenetic agents enhances anti-tumor activity of T cells by upregulating the ACRBP expression in hepatocellular carcinoma. Am J Transl Res., 2021, 137): 7591-7609. DOI:

[19] Bachman J. Reverse-transcription PCR (RT‒PCR). In: Laboratory Methods in enzymology: RNA. Amsterdam: Elsevier, 2013:67–74.

[20] Sipos D, Raposa BL, Freihat O, et al. . Glioblastoma: clinical presentation, multidisciplinary management, and long-term outcomes. Cancers (Basel)., 2025, 17(1): 146

[21] GBD 2016 Brain and Other CNS Cancer Collaborators. Global, regional, and national burden of brain and other CNS cancer, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol., 2019, 18(4): 376-393.

[22] Wei X, Zhang QM, Liu C, et al. . Microrna-1224-5p is a potential prognostic and therapeutic biomarker in glioblastoma: integrating bioinformatics and clinical analyses. Curr Med Sci., 2022, 42(3): 584-596.

[23] Lu HT, Chen YY, Yu YJ, et al. . Gene expression profile identifies LncRNA AL355974.3 as a potential glioma biomarker. Curr Med Sci., 2024, 44(5): 1047-1057.

[24] Bonnal SC, López-Oreja I, Valcárcel J. Roles and mechanisms of alternative splicing in cancer—implications for care. Nat Rev Clin Oncol., 2020, 17(8): 457-474.

[25] Liu WR, Li CY, Xu WH, et al. . Genome-wide analyses of the prognosis-related mRNA alternative splicing landscape and novel splicing factors based on large-scale low grade glioma cohort. Aging., 2020, 12(13): 13684-13700.

[26] Naik A, Lattab B, Qasem H, et al. . Cancer testis antigens: Emerging therapeutic targets leveraging genomic instability in cancer. Mol Ther Oncol., 2024, 32(1200768

[27] Robbins PF, Morgan RA, Feldman SA, et al. . Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol., 2011, 29(7): 917-924.

[28] Rapoport AP, Stadtmauer EA, Binder-Scholl GK, et al. . NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat Med., 2015, 21(8): 914-921.

[29] Eifuku R, Takenoyama M, Yoshino I, et al. . Analysis of MAGE-3 derived synthetic peptide as a human lung cancer antigen recognized by cytotoxic T lymphocytes. Int J Clin Oncol., 2001, 6(1): 34-39.

[30] Kanemori Y, Ryu JH, Sudo M, Niida-Araida Y, Kodaira K, Takenaka M, Kohno N, Sugiura S, Kashiwabara S, Baba T. Two functional forms of ACRBP/sp32 are produced by pre-mRNA alternative splicing in the mouse. Biol Reprod., 2013, 88(4): 105

[31] Kanemori Y, Koga Y, Sudo M, et al. . Biogenesis of sperm acrosome is regulated by pre-mRNA alternative splicing of Acrbp in the mouse. Proc Natl Acad Sci U S A., 2016, 11326): E3696-E3705.

[32] Shao Y, Chong W, Liu X, et al. . Alternative splicing-derived intersectin1-L and intersectin1-S exert opposite function in glioma progression. Cell Death Dis., 2019, 10(6): 431

[33] Ben Mrid R, El Guendouzi S, Mineo M, et al. . The emerging roles of aberrant alternative splicing in glioma. Cell Death Discov., 2025, 11(1): 50

[34] Louis DN, Perry A, Wesseling P, et al. . The 2021 WHO classification of tumors of the central nervous system: a summary. Neuro Oncol., 2021, 23(8): 1231-1251.

[35] Weller M, Wen PY, Chang SM, et al. . Glioma. Nat Rev Dis Primers., 2024, 10: 33

[36] Frappaz D, Bonneville-Levard A, Ricard D, et al. . Assessment of Karnofsky (KPS) and WHO (WHO-PS) performance scores in brain tumour patients: the role of clinician bias. Support Care Cancer., 2021, 29(4): 1883-1891. DOI:

[37] Kim TK, Eberwine JH. Mammalian cell transfection: the present and the future. Anal Bioanal Chem., 2010, 397(8): 3173-3178.

[38] Pluta K, Kacprzak MM. Use of HIV as a gene transfer vector. Acta Biochim Pol., 2009, 56(4): 531-595.

[39] Fuchs A, Riegler S, Ayatollahi Z, et al. . Targeting alternative splicing by RNAi: from the differential impact on splice variants to triggering artificial pre-mRNA splicing. Nucleic Acids Res., 2021, 49(2): 1133-1151.

[40] Schertzer MD, Stirn A, Isaev K, et al. . Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox. Nat Commun., 2025, 16(1): 6948

[41] Zhou X, Li X, Yu L, et al. . The RNA-binding protein SRSF1 is a key cell cycle regulator via stabilizing NEAT1 in glioma. Int J Biochem Cell Biol., 2019, 113: 75-86.

[42] Fuentes-Fayos AC, Vázquez-Borrego MC, Jiménez-Vacas JM, et al. . Splicing machinery dysregulation drives glioblastoma development/aggressiveness: oncogenic role of SRSF3. Brain., 2020, 143(11): 3273-3293.

[43] Bhardwaj S, Sanjay, Yadav AK. Higher isoform of hnRNPA1 confer Temozolomide resistance in U87MG & LN229 glioma cells. J Neurooncol. 2025;171(1):47–63.

Funding

Innovation Project of Guangxi Graduate Education(YCSW2025248)

Natural Science Foundation of Guangxi Province(Grant No: 2023GXNSFAA026120)

the National Natural Science Foundation of China(Grant No. 82260608)

the Key Laboratory of Basic Research on Regional Diseases (Guangxi Medical University), Education Department of Guangxi Zhuang Autonomous Region(Grant No. RTFY202302)

RIGHTS & PERMISSIONS

The Author(s), under exclusive licence to the Huazhong University of Science and Technology

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