2. Academic Validation
  3. Daidzein reprograms EP300/CREBBP-deficient immune evasion via targeting the PPARγ-ANGPT4/Tie2 axis in hypopharyngeal squamous cell carcinoma

Daidzein reprograms EP300/CREBBP-deficient immune evasion via targeting the PPARγ-ANGPT4/Tie2 axis in hypopharyngeal squamous cell carcinoma

  • Phytomedicine. 2025 Dec:149:157488. doi: 10.1016/j.phymed.2025.157488.
Wei Mao 1,  Chen Feng 2,  Gang Gao 3,  Biwen Jiao 3,  Peiyan Zhu 3,  Yufan Chen 4,  Yongzhi Men 5,  Pin Dong 6,  Baoxin Wang 7
Affiliations

Affiliations

  • 1 Department of Otolaryngology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 85#Wujin Road, Hongkou, Shanghai 200080, China; Department of Otolaryngology, Head and Neck Surgery, Qilu Hospital of Shandong University, NHC Key Laboratory of Otorhinolaryngology (Shandong University), Jinan, China; Eye Institute of Shandong First Medical University, Eye Hospital of Shandong First Medical University(Shandong Eye Hospital), China. Electronic address: [email protected].
  • 2 Department of Otolaryngology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 85#Wujin Road, Hongkou, Shanghai 200080, China; Department of Otolaryngology, Head and Neck Surgery, Qilu Hospital of Shandong University, NHC Key Laboratory of Otorhinolaryngology (Shandong University), Jinan, China; Eye Institute of Shandong First Medical University, Eye Hospital of Shandong First Medical University(Shandong Eye Hospital), China. Electronic address: [email protected].
  • 3 Department of Otolaryngology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 85#Wujin Road, Hongkou, Shanghai 200080, China; Department of Otolaryngology, Head and Neck Surgery, Qilu Hospital of Shandong University, NHC Key Laboratory of Otorhinolaryngology (Shandong University), Jinan, China; Eye Institute of Shandong First Medical University, Eye Hospital of Shandong First Medical University(Shandong Eye Hospital), China.
  • 4 Department of Otolaryngology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 85#Wujin Road, Hongkou, Shanghai 200080, China; Department of Otolaryngology, Head and Neck Surgery, Qilu Hospital of Shandong University, NHC Key Laboratory of Otorhinolaryngology (Shandong University), Jinan, China; Eye Institute of Shandong First Medical University, Eye Hospital of Shandong First Medical University(Shandong Eye Hospital), China. Electronic address: [email protected].
  • 5 Department of Otolaryngology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 85#Wujin Road, Hongkou, Shanghai 200080, China; Department of Otolaryngology, Head and Neck Surgery, Qilu Hospital of Shandong University, NHC Key Laboratory of Otorhinolaryngology (Shandong University), Jinan, China; Eye Institute of Shandong First Medical University, Eye Hospital of Shandong First Medical University(Shandong Eye Hospital), China. Electronic address: [email protected].
  • 6 Department of Otolaryngology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 85#Wujin Road, Hongkou, Shanghai 200080, China; Department of Otolaryngology, Head and Neck Surgery, Qilu Hospital of Shandong University, NHC Key Laboratory of Otorhinolaryngology (Shandong University), Jinan, China; Eye Institute of Shandong First Medical University, Eye Hospital of Shandong First Medical University(Shandong Eye Hospital), China. Electronic address: [email protected].
  • 7 Department of Otolaryngology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, 85#Wujin Road, Hongkou, Shanghai 200080, China; Department of Otolaryngology, Head and Neck Surgery, Qilu Hospital of Shandong University, NHC Key Laboratory of Otorhinolaryngology (Shandong University), Jinan, China; Eye Institute of Shandong First Medical University, Eye Hospital of Shandong First Medical University(Shandong Eye Hospital), China. Electronic address: [email protected].
PMID: 41240538 DOI: 10.1016/j.phymed.2025.157488
Abstract

Background: Hypopharyngeal squamous cell carcinoma (HPSCC) is a lethal malignancy with limited treatment options and poor survival rates. Recent studies have revealed that mutations in the histone acetyltransferases EP300 and CREBBP drive tumor progression by disrupting the chromatin structure and impairing antitumor immunity. However, no targeted therapies are currently available to treat these epigenetic defects. Natural compounds with epigenetic regulatory potential offer a promising therapeutic avenue but remain largely unexplored in HPSCC.

Methods: Whole-exome and transcriptomic Sequencing (WES) were conducted on HPSCC tissues to identify driver mutations and downstream transcriptional alterations. Histone acetylation profiles were analyzed by mass spectrometry and confirmed by western blotting. The effects of EP300/CREBBP mutations on H3K27ac enrichment at the PPARγ promoter were validated by ChIP-qPCR. Candidate targets, including PPARγ and ANGPT4, were identified through bioinformatics screening and verified by qRT-PCR and immunoblotting. Stable HPSCC cell lines carrying EP300 or CREBBP mutations were established via lentiviral transduction. Cell proliferation, Apoptosis, and invasion were examined using Cell Counting Kit-8, TUNEL staining, and Transwell assays. The transcriptional regulation of ANGPT4 by PPARγ was evaluated using a dual-luciferase reporter assay. CD4⁺ T-cell subsets were analyzed by spectral flow cytometry. A xenograft model was established with FaDu cells in nude mice, and daidzein was administered intraperitoneally. Tumor growth, Ki-67 immunohistochemistry, and ELISA-based cytokine detection were used to assess therapeutic efficacy.

Results: Mutations in EP300 and CREBBP caused a marked loss of H3K27 acetylation and suppression of PPARγ, which in turn activated the ANGPT4/Tie2 oncogenic pathway and reshaped the immune microenvironment toward a regulatory T-cell-dominant profile. Treatment with daidzein effectively restored histone acetylation and PPARγ expression, leading to the inhibition of ANGPT4/Tie2 signaling and reversal of tumor-promoting phenotypes. In cultured HPSCC cells, daidzein reduced proliferation and invasion while inducing pronounced apoptotic changes. Dual-luciferase assays confirmed that PPARγ directly transactivated the ANGPT4 promoter, providing mechanistic evidence for its regulatory role. In animal models, intraperitoneal administration of daidzein (20-40 mg/kg) markedly delayed tumor growth, lowered Ki-67 expression, and reduced serum levels of immunosuppressive cytokines such as TGF-β and IL-35. Collectively, these findings indicate that daidzein acts as a dual activator of EP300 and PPARγ, re-establishing epigenetic balance and restoring antitumor immunity in EP300/CREBBP-deficient HPSCC.

Conclusions: Through an integrated multi-omics strategy combining whole-exome Sequencing, transcriptome profiling, and histone modification analysis in a Chinese HPSCC cohort, we identified recurrent loss-of-function mutations in EP300/CREBBP. These mutations reduced H3K27 acetylation and downregulated PPARγ, thereby activating the ANGPT4/Tie2 oncogenic signaling axis. Functional assays confirmed enhanced proliferation, invasion, and immune evasion, characterized by a regulatory T cell-dominant immune phenotype. Treatment with daidzein restored PPARγ expression, suppressed ANGPT4/Tie2 signaling, and reversed these malignant features, both in vitro and in vivo. Compared with previous studies, our work not only elucidates the functional consequences of EP300/CREBBP mutations in HPSCC but also proposes a novel therapeutic strategy targeting this axis. Importantly, we reveal a previously unrecognized EP300/CREBBP-PPARγ-ANGPT4/Tie2 axis and identify daidzein as a dual agonist of EP300 and PPARγ, providing mechanistic insights and translational potential for HPSCC therapy.

Keywords

Daidzein; EP300/CREBBP mutations; HPSCC; Histone acetylation; PPARγ; Regulatory T cells.

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