{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,7]],"date-time":"2025-12-07T05:23:58Z","timestamp":1765085038809,"version":"3.46.0"},"reference-count":38,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2025,12,5]],"date-time":"2025-12-05T00:00:00Z","timestamp":1764892800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["82260608","82260554"],"award-info":[{"award-number":["82260608","82260554"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Natural Science Foundation of Guangxi Province","award":["2022GXNSFAA035639","2018GXNSFAA050058"],"award-info":[{"award-number":["2022GXNSFAA035639","2018GXNSFAA050058"]}]},{"name":"Guangxi Young and Middle-aged University Teachers\u2019 Scientific Research Ability Enhancement Project","award":["2025KY0129"],"award-info":[{"award-number":["2025KY0129"]}]},{"DOI":"10.13039\/501100012543","name":"Youth Science Foundation of Guangxi Medical University","doi-asserted-by":"crossref","award":["GXMUYSF202536"],"award-info":[{"award-number":["GXMUYSF202536"]}],"id":[{"id":"10.13039\/501100012543","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Innovation and Entrepreneurship Training Program Project for College Students in Guangxi Medical University","award":["201610598011","201910598054"],"award-info":[{"award-number":["201610598011","201910598054"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Intratumoral heterogeneity poses significant challenges to the efficacy of cancer immunotherapy. Melanoma-associated antigen D4 (MAGED4) has been proposed as a potential immunotherapeutic target in oral squamous cell carcinoma (OSCC). This study aims to investigate the expression of MAGED4, focusing on its intratumoral expression heterogeneity and the underlying epigenetic regulation mechanism. Utilizing public online databases, immunohistochemical analyses of clinical specimens, and single-cell RNA sequencing data, we found that MAGED4 was overexpressed with significant intratumoral heterogeneity in OSCC tissues. Methylation-promoter luciferase reporter assays revealed that MAGED4 transcription was suppressed by DNA methylation at its promoter region. Additionally, co-expression analysis implicated a potential role for histone acetylation in regulating MAGED4. To functionally validate these findings, we treated OSCC cells with the DNA methyltransferase inhibitor 5-aza-2\u2032-deoxycytidine (DAC) and histone deacetylase inhibitors trichostatin A (TSA) and valproic acid (VPA). The triple-drug combination treatment resulted in the most robust reactivation of MAGED4 expression, correlating with promoter DNA demethylation and enhanced acetylation of H3K9 and H3K27 at the MAGED4 promoter. Our findings elucidate critical epigenetic mechanisms contributing to MAGED4 heterogeneity in OSCC and highlight the potential of combination epigenetic therapies to reverse this heterogeneity, thereby providing a foundation for exploring such approaches to improve immunotherapeutic outcomes.<\/jats:p>","DOI":"10.3390\/ijms262411772","type":"journal-article","created":{"date-parts":[[2025,12,5]],"date-time":"2025-12-05T10:50:38Z","timestamp":1764931838000},"page":"11772","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Intratumoral Heterogeneity of MAGED4 Expression in Oral Squamous Cell Carcinoma: Epigenetic Mechanisms and Therapeutic Implications"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-6787-7630","authenticated-orcid":false,"given":"Huan","family":"Xie","sequence":"first","affiliation":[{"name":"Department of Histology and Embryology, School of Basic Medicine Science, Guangxi Medical University, Nanning 530021, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Feng","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Histology and Embryology, School of Basic Medicine Science, Guangxi Medical University, Nanning 530021, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaoqiong","family":"Zou","sequence":"additional","affiliation":[{"name":"Department of Histology and Embryology, School of Basic Medicine Science, Guangxi Medical University, Nanning 530021, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaoqing","family":"Yu","sequence":"additional","affiliation":[{"name":"Department of Histology and Embryology, School of Basic Medicine Science, Guangxi Medical University, Nanning 530021, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sheng","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Histology and Embryology, School of Basic Medicine Science, Guangxi Medical University, Nanning 530021, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanjing","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Histology and Embryology, School of Basic Medicine Science, Guangxi Medical University, Nanning 530021, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Weixia","family":"Nong","sequence":"additional","affiliation":[{"name":"Department of Histology and Embryology, School of Basic Medicine Science, Guangxi Medical University, Nanning 530021, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Limin","family":"Xie","sequence":"additional","affiliation":[{"name":"General Practice School, Guangxi Medical University, Nanning 530021, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yi","family":"Wang","sequence":"additional","affiliation":[{"name":"General Practice School, Guangxi Medical University, Nanning 530021, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bin","family":"Luo","sequence":"additional","affiliation":[{"name":"Department of Histology and Embryology, School of Basic Medicine Science, Guangxi Medical University, Nanning 530021, China"},{"name":"Key Laboratory of Basic Research on Regional Diseases (Guangxi Medical University), Education Department of Guangxi Zhuang Autonomous Region, Nanning 530021, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaoxun","family":"Xie","sequence":"additional","affiliation":[{"name":"Department of Histology and Embryology, School of Basic Medicine Science, Guangxi Medical University, Nanning 530021, China"},{"name":"Key Laboratory of Basic Research on Regional Diseases (Guangxi Medical University), Education Department of Guangxi Zhuang Autonomous Region, Nanning 530021, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qingmei","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Histology and Embryology, School of Basic Medicine Science, Guangxi Medical University, Nanning 530021, China"},{"name":"Key Laboratory of Basic Research on Regional Diseases (Guangxi Medical University), Education Department of Guangxi Zhuang Autonomous Region, Nanning 530021, China"},{"name":"Guangxi Health Commission Key Laboratory of Basic Research on Brain Function and Disease, Nanning 530021, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Chen, Y., Wei, Y., Yang, C., Liu, C.-J., Yeh, L.-Y., Chou, C.-H., Chang, K.-W., and Lin, S.-C. (2019). miR-125b suppresses oral oncogenicity by targeting the anti-oxidative gene PRXL2A. Redox Biol., 22.","DOI":"10.1016\/j.redox.2019.101140"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1186\/s13046-017-0553-x","article-title":"The role of NLRP3 inflammasome in 5-fluorouracil resistance of oral squamous cell carcinoma","volume":"36","author":"Feng","year":"2017","journal-title":"J. Exp. Clin. Cancer Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2544","DOI":"10.1002\/hep.31921","article-title":"Lenvatinib Targets FGF Receptor 4 to Enhance Antitumor Immune Response of Anti-Programmed Cell Death-1 in HCC","volume":"74","author":"Yi","year":"2021","journal-title":"Hepatology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e25895","DOI":"10.1016\/j.heliyon.2024.e25895","article-title":"Molecular basis, potential biomarkers, and future prospects of OSCC and PD-1\/PD-L1 related immunotherapy methods","volume":"10","author":"Cheng","year":"2024","journal-title":"Heliyon"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1002\/jso.21580","article-title":"Differential gene expression profile of MAGE family in taiwanese patients with colorectal cancer","volume":"102","author":"Chung","year":"2010","journal-title":"J. Surg. Oncol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1251","DOI":"10.1007\/s00262-015-1731-3","article-title":"A phase I trial combining decitabine\/dendritic cell vaccine targeting MAGE-A1, MAGE-A3 and NY-ESO-1 for children with relapsed or therapy-refractory neuroblastoma and sarcoma","volume":"64","author":"Krishnadas","year":"2015","journal-title":"Cancer Immunol. Immunother."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1131","DOI":"10.3892\/or.2012.1956","article-title":"Dendritic cell-based vaccination in metastatic melanoma patients: Phase II clinical trial","volume":"28","author":"Oshita","year":"2012","journal-title":"Oncol. Rep."},{"key":"ref_8","first-page":"4809","article-title":"MAGE-E1, a new member of the melanoma-associated antigen gene family and its expression in human glioma","volume":"61","author":"Sasaki","year":"2001","journal-title":"Cancer Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"3495","DOI":"10.7314\/APJCP.2014.15.8.3495","article-title":"MAGED4 expression in glioma and upregulation in glioma cell lines with 5-aza-2\u2032-deoxycytidine treatment","volume":"15","author":"Zhang","year":"2014","journal-title":"Asian Pac. J. Cancer Prev."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.canlet.2012.03.025","article-title":"Over-expression of MAGED4B increases cell migration and growth in oral squamous cell carcinoma and is associated with poor disease outcome","volume":"321","author":"Chong","year":"2012","journal-title":"Cancer Lett."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"712","DOI":"10.1016\/j.oraloncology.2008.11.002","article-title":"Gene expression in human oral squamous cell carcinoma is influenced by risk factor exposure","volume":"45","author":"Cheong","year":"2009","journal-title":"Oral Oncol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3214","DOI":"10.4161\/hv.29226","article-title":"Identification of immunogenic MAGED4B peptides for vaccine development in oral cancer immunotherapy","volume":"10","author":"Lim","year":"2014","journal-title":"Hum. Vaccine Immunother."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1080\/21645515.2018.1520584","article-title":"In vitro evaluation of dual-antigenic PV1 peptide vaccine in head and neck cancer patients","volume":"15","author":"Chai","year":"2019","journal-title":"Hum. Vaccine Immunother."},{"key":"ref_14","first-page":"3918","article-title":"Overexpression of MAGE-D4 in colorectal cancer is a potentially prognostic biomarker and immunotherapy target","volume":"7","author":"Zhang","year":"2014","journal-title":"Int. J. Clin. Exp. Pathol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Bi, S., Zhang, Q., Zeng, X., Liu, C., Nong, W.-X., Xie, H., Li, F., Lin, L.-N., Luo, B., and Ge, Y.-Y. (2022). Combined treatment with epigenetic agents enhances anti-tumor activity of MAGE-D4 peptide-specific T cells by upregulating the MAGE-D4 expression in glioma. Front. Oncol., 12.","DOI":"10.3389\/fonc.2022.873639"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"17588359231170474","DOI":"10.1177\/17588359231170474","article-title":"The new era of prostate-specific membrane antigen-directed immunotherapies and beyond in advanced prostate cancer: A review","volume":"15","author":"Martin","year":"2023","journal-title":"Ther. Adv. Med. Oncol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1158\/1541-7786.MCR-05-0229","article-title":"Promoter demethylation and histone acetylation mediate gene expression of MAGE-A1, -A2, -A3, and -A12 in human cancer cells","volume":"4","author":"Wischnewski","year":"2006","journal-title":"Mol. Cancer Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"618","DOI":"10.3109\/14653249.2010.529893","article-title":"Epigenetic modulation of MAGE-A3 antigen expression in multiple myeloma following treatment with the demethylation agent 5-azacitidine and the histone deacetlyase inhibitor MGCD0103","volume":"13","author":"Szmania","year":"2011","journal-title":"Cytotherapy"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Binder, H., Schmidt, M., Hopp, L., Davitavyan, S., Arakelyan, A., and Loeffler-Wirth, H. (2022). Integrated Multi-Omics Maps of Lower-Grade Gliomas. Cancers, 14.","DOI":"10.3390\/cancers14112797"},{"key":"ref_20","first-page":"2241","article-title":"Synergistic regulation of methylation and SP1 on MAGE-D4 transcription in glioma","volume":"13","author":"Liu","year":"2021","journal-title":"Am. J. Transl. Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e2405332","DOI":"10.1002\/advs.202405332","article-title":"SNORA28 Promotes Proliferation and Radioresistance in Colorectal Cancer Cells through the STAT3 Pathway by Increasing H3K9 Acetylation in the LIFR Promoter","volume":"11","author":"Liu","year":"2024","journal-title":"Adv. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e2408049121","DOI":"10.1073\/pnas.2408049121","article-title":"ACSL4-mediated H3K9 and H3K27 hyperacetylation upregulates SNAIL to drive TNBC metastasis","volume":"121","author":"Sinha","year":"2024","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1038\/s41467-023-44540-0","article-title":"MCT1-governed pyruvate metabolism is essential for antibody class-switch recombination through H3K27 acetylation","volume":"15","author":"Chi","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"214","DOI":"10.1016\/j.cllc.2011.09.007","article-title":"Characterization of melanoma-associated antigen-a genes family differential expression in non-small-cell lung cancers","volume":"13","author":"Karimi","year":"2012","journal-title":"Clin. Lung Cancer"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Curioni-Fontecedro, A., Pitocco, R., Schoenewolf, N.L., Holzmann, D., Soldini, D., Dummer, R., Calvieri, S., Moch, H., Mihic-Probst, D., and Fitsche, A. (2015). Intratumoral Heterogeneity of MAGE-C1\/CT7 and MAGE-C2\/CT10 Expression in Mucosal Melanoma. BioMed Res. Int., 2015, Erratum in BioMed Res. Int.\u00a02019, 2019, 5256364.","DOI":"10.1155\/2019\/5256364"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1016\/j.ctrv.2018.04.009","article-title":"MAGE-A antigens as targets for cancer immunotherapy","volume":"67","author":"Schooten","year":"2018","journal-title":"Cancer Treat. Rev."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1186\/s13148-018-0550-8","article-title":"Epigenetic regulation of MAGE family in human cancer progression-DNA methylation, histone modification, and non-coding RNAs","volume":"10","author":"Lian","year":"2018","journal-title":"Clin. Epigenetics"},{"key":"ref_28","first-page":"105","article-title":"Tumor heterogeneity: Causes and consequences","volume":"1805","author":"Marusyk","year":"2010","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1047","DOI":"10.1093\/jnci\/djz238","article-title":"Oral Leukoplakia and Risk of Progression to Oral Cancer: A Population-Based Cohort Study","volume":"112","author":"Chaturvedi","year":"2020","journal-title":"J. Natl. Cancer Inst."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3293","DOI":"10.1200\/JCO.2015.61.1509","article-title":"Immunology and Immunotherapy of Head and Neck Cancer","volume":"33","author":"Ferris","year":"2015","journal-title":"J. Clin. Oncol."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Geum, D.H., Hwang, D.S., Lee, C.H., Cho, S.D., Jang, M.A., Ryu, M.H., and Kim, U.K. (2022). PD-L1 Expression Correlated with Clinicopatho-logical Factors and Akt\/Stat3 Pathway in Oral SCC. Life, 12.","DOI":"10.3390\/life12020238"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e162907","DOI":"10.1172\/jci.insight.162907","article-title":"Reversible epigenetic alterations mediate PSMA expression heterogeneity in advanced metastatic prostate cancer","volume":"8","author":"Sayar","year":"2023","journal-title":"JCI Insight"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1186\/s13058-016-0685-5","article-title":"An integrated genomics analysis of epigenetic subtypes in human breast tumors links DNA methylation patterns to chromatin states in normal mammary cells","volume":"18","author":"Holm","year":"2016","journal-title":"Breast Cancer Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"653","DOI":"10.1038\/ni.3148","article-title":"TET1 is a tumor suppressor of hematopoietic malignancy","volume":"16","author":"Cimmino","year":"2015","journal-title":"Nat. Immunol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1186\/s13148-020-00835-3","article-title":"Crosstalk between DNA methylation and histone acetylation triggers GDNF high transcription in glioblastoma cells","volume":"12","author":"Zhang","year":"2020","journal-title":"Clin. Epigenetics"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1016\/0092-8674(92)90610-O","article-title":"Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA","volume":"69","author":"Lewis","year":"1992","journal-title":"Cell"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"386","DOI":"10.1038\/30764","article-title":"Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex","volume":"393","author":"Nan","year":"1998","journal-title":"Nature"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"D1425","DOI":"10.1093\/nar\/gkac959","article-title":"TISCH2: Expanded datasets and new tools for single-cell transcriptome analyses of the tumor microen-vironment","volume":"51","author":"Han","year":"2023","journal-title":"Nucleic Acids Res."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/24\/11772\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,7]],"date-time":"2025-12-07T05:21:22Z","timestamp":1765084882000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/24\/11772"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,5]]},"references-count":38,"journal-issue":{"issue":"24","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["ijms262411772"],"URL":"https:\/\/doi.org\/10.3390\/ijms262411772","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2025,12,5]]}}}