Journal of Shanghai Jiao Tong University (Medical Science) >

2026 , Vol. 46 >Issue 2: 151 - 162

DOI: https://doi.org/10.3969/j.issn.1674-8115.2026.02.003

Basic research

Study on how histone lysine lactylation regulates zinc finger protein 668 to facilitate proliferation of head and neck squamous cell carcinoma cells

  • Yin Wenyu ,
  • Wang Ruijie ,
  • Ma Hailong ,
  • Jin Shufang
Expand
  • 1.School of Stomatology, Binzhou Medical University, Yantai 264003, China
    2.Department of Second Dental Center, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology; Shanghai Research Institute of Stomatology, Shanghai 200011, China
    3.Department of Oral Maxillofacial & Head and Neck Oncology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology; Shanghai Research Institute of Stomatology, Shanghai 200011, China
Jin Shufang, E-mail: 72300120147@shsmu.edu.cn.
Ma Hailong, E-mail: mahl21@sjtu.edu.cn.

Received date: 2025-07-21

  Accepted date: 2025-11-10

  Online published: 2026-02-28

Supported by

National Natural Science Foundation of China(82172897,82272983);Shanghai Rising-Star Program(22QA1405300);Natural Science Foundation of Shanghai(22ZR1436800);Fund for Excellent Young Scholars of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine(JYYQ010)

Fold

Abstract

Objective ·To investigate the regulation of zinc finger protein 668 (ZNF668) expression in head and neck squamous cell carcinoma (HNSCC) by histone lysine lactylation (Kla), and to elucidate the molecular mechanisms by which ZNF668 promotes the proliferation of HNSCC cells. Methods ·Multiplex immunofluorescence was used to detect pan-lysine lactylation (PKla) levels in tissue microarrays (TMAs) constructed from HNSCC and adjacent normal oral mucosal tissues, and the correlation between PKla expression and clinicopathological characteristics was analyzed. Western blotting was performed to identify specific sites where exogenous lactate treatment regulated Kla in HNSCC cells. CUT&Tag technology was utilized to screen histone lactylation-related and open chromatin genes. These data were combined with RNA sequencing (RNA-seq) experiments to detect differentially expressed downstream genes stimulated by lactate at the transcriptome level, and to screen specific downstream target genes regulated by Kla. Chromatin immunoprecipitation (ChIP) assays were used to verify the accessibility of Kla to downstream target genes. Western blotting was used to analyze the expression levels of ZNF668 in human normal oral keratinocytes (NOK) and HNSCC cells. The expression of ZNF668 mRNA in NOK and HNSCC cells was verified by quantitative real-time polymerase chain reaction (qRT-PCR). Western blotting was used to detect the expression of the most sensitive modification site to lactate stimulation and ZNF668 under control treatments with or without lactate. The 5-ethynyl-2'-deoxyuridine (EdU) and cell counting kit-8 (CCK-8) assays were used to analyze the effects of knocking down ZNF668 on the proliferation ability of HNSCC cells. Western blotting was used to analyze the expression of WNT3A (wingless-type MMTV integration site family member 3A), β-catenin, and G1/S-specific cyclin-D1 (CCND1) proteins after knocking down ZNF668. Immunohistochemistry (IHC) was used to detect the expression of ZNF668 and cell proliferation in the subcutaneous tumors of nude mice stimulated by lactate. Results ·Multiplex immunofluorescence results indicated that the level of PKla modification in HNSCC tissues was higher than that in normal oral mucosa, and that a high level of lactylation modification was associated with advanced TNM stage. Western blotting results showed that lactate promoted Kla in HNSCC cells, and the Kla at the H3K9 site was the most significant. The results of CUT&Tag combined with RNA-seq screening and ChIP-PCR verification demonstrated that ZNF668 was one of the main downstream targets of H3K9la. Western blotting and qRT-PCR analyses showed that the expression level ofZNF668 in HNSCC cells was significantly higher than that in NOK cells, and that the expression of H3K9la and ZNF668 in HNSCC cells was positively correlated with lactate stimulation. After knocking down ZNF668 in HNSCC cells, the proliferative ability of the cells was significantly reduced compared to the control group. Meanwhile, the protein expressions of WNT3A, β-catenin, and CCND1 also showed a downward trend. The IHC assay demonstrated that lactate stimulation increased the expression of ZNF668 and promoted cell proliferation in subcutaneous tumors of nude mice. Conclusion ·Histone lysine lactylation promotes the expression of ZNF668, which plays a significant cancer-promoting role in HNSCC. ZNF668 can further regulate the proliferation of tumor cells by enhancing the expression level of the WNT3A/β-catenin/CCND1 signaling axis.

Key words： head and neck squamous cell carcinoma (HNSCC); histone lactylation modification (Kla); zinc finger protein 668 (ZNF668); cell proliferation

Cite this article

Yin Wenyu , Wang Ruijie , Ma Hailong , Jin Shufang . Study on how histone lysine lactylation regulates zinc finger protein 668 to facilitate proliferation of head and neck squamous cell carcinoma cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026 , 46(2) : 151 -162 . DOI: 10.3969/j.issn.1674-8115.2026.02.003

References

[1] Chow L Q M. Head and neck cancer[J]. N Engl J Med, 2020, 382(1): 60-72.
[2] Han B F, Zheng R S, Zeng H M, et al. Cancer incidence and mortality in China, 2022[J]. J Natl Cancer Cent, 2024, 4(1): 47-53.
[3] Sun Z, Sun X D, Chen Z W, et al. Head and neck squamous cell carcinoma: risk factors, molecular alterations, immunology and peptide vaccines[J]. Int J Pept Res Ther, 2022, 28(1): 19.
[4] 郑荣寿, 陈茹, 韩冰峰, 等. 2022年中国恶性肿瘤流行情况分析[J]. 中华肿瘤杂志, 2024, 46(3): 221-231.
  Zheng R S, Chen R, Han B F, et al. Cancer incidence and mortality in China, 2022[J]. Chinese Journal of Oncology, 2024, 46(3): 221-231.
[5] Dai Z W, Ramesh V, Locasale J W. The evolving metabolic landscape of chromatin biology and epigenetics[J]. Nat Rev Genet, 2020, 21(12): 737-753.
[6] Yang Z J, Yan C, Ma J Q, et al. Lactylome analysis suggests lactylation-dependent mechanisms of metabolic adaptation in hepatocellular carcinoma[J]. Nat Metab, 2023, 5(1): 61-79.
[7] Zhong X Y, He X F, Wang Y X, et al. Warburg effect in colorectal cancer: the emerging roles in tumor microenvironment and therapeutic implications[J]. J Hematol Oncol, 2022, 15(1): 160.
[8] 陈燕, 胡泽楠, 姜雷. 乳酸对肿瘤免疫微环境及免疫治疗影响的研究进展[J]. 中国肿瘤临床, 2025, 52(8): 407-412.
  Chen Y, Hu Z N, Jiang L. Research progress on the effects of lactic acid on tumor immune microenvironment and immunotherapy[J]. Chinese Journal of Clinical Oncology, 2025, 52(8): 407-412.
[9] Yang K, Fan M, Wang X H, et al. Lactate promotes macrophage HMGB1 lactylation, acetylation, and exosomal release in polymicrobial sepsis[J]. Cell Death Differ, 2022, 29(1): 133-146.
[10] 斯航, 冯燕, 余丽. 锌指蛋白在口腔癌发生发展中的作用研究进展[J]. 口腔疾病防治, 2025(5): 425-432.
  Si H, Feng Y, Yu L. Research progress on the role of zinc finger proteins in the development of oral cancer[J]. Journal of Dental Prevention & Treatment, 2025(5): 425-432.
[11] Iyer A S, Shaik M R, Raufman J P, et al. The roles of zinc finger proteins in colorectal cancer[J]. Int J Mol Sci, 2023, 24(12): 10249.
[12] Wei C, Gao Y J, Chen X T, et al. ZNF668: a new diagnostic predictor of kidney renal clear cell carcinoma[J]. Anticancer Drugs, 2022, 33(1): e491-e499.
[13] 李立方, 仇丽, 胡凯. 锌指蛋白在基因转录翻译及肿瘤发生发展中的调控作用机制研究进展[J]. 山东医药, 2021, 61(30): 83-87.
  Li L F, Qiu L, Hu K. Research progress on the regulatory mechanism of zinc finger protein in gene transcription and translation and tumor occurrence and development[J]. Shandong Medical Journal, 2021, 61(30): 83-87.
[14] Chen Y P, Wu J H, Zhai L H, et al. Metabolic regulation of homologous recombination repair by MRE11 lactylation[J]. Cell, 2024, 187(2): 294-311.e21.
[15] Sun Y D, He Q, Li J J, et al. A GSTP1-mediated lactic acid signaling promotes tumorigenesis through the PPP oxidative branch[J]. Cell Death Dis, 2023, 14(7): 463.
[16] Xiong J, He J, Zhu J, et al. Lactylation-driven METTL3-mediated RNA m6A modification promotes immunosuppression of tumor-infiltrating myeloid cells[J]. Mol Cell, 2022, 82(9): 1660-1677.e10.
[17] Deng J, Li Y K, Yin L L, et al. Histone lactylation enhances GCLC expression and thus promotes chemoresistance of colorectal cancer stem cells through inhibiting ferroptosis[J]. Cell Death Dis, 2025, 16(1): 193.
[18] LI T T, HU C H, HUANG T H, et al. Cancer-associated fibroblasts foster a high-lactate microenvironment to drive perineural invasion in pancreatic cancer[J]. Cancer Res, 2025, 85(12): 2199-2217.
[19] Hu X M, Huang Z Q, Li L Y. LDHB mediates histone lactylation to activate PD-L1 and promote ovarian cancer immune escape[J]. Cancer Invest, 2025, 43(1): 70-79.
[20] Pan L H, Feng F, Wu J Q, et al. Demethylzeylasteral targets lactate by inhibiting histone lactylation to suppress the tumorigenicity of liver cancer stem cells[J]. Pharmacol Res, 2022, 181: 106270.
[21] Wang R J, Li C W, Cheng Z Y, et al. H3K9 lactylation in malignant cells facilitates CD8+ T cell dysfunction and poor immunotherapy response[J]. Cell Rep, 2024, 43(9): 114686.
[22] Alsaif H S, Al Ali H, Faqeih E, et al. ZNF668 deficiency causes a recognizable disorder of DNA damage repair[J]. Hum Genet, 2021, 140(9): 1395-1401.
[23] Hu R Z, Wang E, Peng G, et al. Zinc finger protein 668 interacts with Tip60 to promote H2AX acetylation after DNA damage[J]. Cell Cycle, 2013, 12(13): 2033-2041.
[24] Lin Z W, Liu J K. lncRNA DQ786243 promotes hepatocellular carcinoma cell invasion and proliferation by regulating the miR-15b-5p/Wnt3A axis[J]. Mol Med Rep, 2021, 23(5): 318.
[25] Zhang B C, Hou M H, Huang J Y, et al. Pax6 regulates neuronal migration and cell proliferation via interacting with Wnt3a during cortical development[J]. Sci Rep, 2025, 15(1): 4726.
[26] Jia X X, Wu B, Huang J H, et al. YAP and Wnt3a independently promote AECIIs proliferation and differentiation by increasing nuclear β-catenin expression in experimental bronchopulmonary dysplasia[J]. Int J Mol Med, 2021, 47(1): 195-206.
[27] Wang J, Shen D X, Li S C, et al. LINC00665 activating Wnt3a/β- catenin signaling by bond with YBX1 promotes gastric cancer proliferation and metastasis[J]. Cancer Gene Ther, 2023, 30(11): 1530-1542.
[28] Chen L, Huang X F, Li X C, et al. Geniposide promotes the proliferation and differentiation of MC3T3-E1 and ATDC5 cells by regulation of microRNA-214[J]. Int Immunopharmacol, 2020, 80: 106121.
[29] Wei Y J, Chen W, Li Z H, et al. EIF3H stabilizes CCND1 to promotes intrahepatic cholangiocarcinoma progression via Wnt/β- catenin signaling[J]. FASEB J, 2022, 36(12): e22647.
[30] Wu Q B, Gao X Y, Lin Y F, et al. Integrating epigenetics, proteomics, and metabolomics to reveal the involvement of Wnt/β-catenin signaling pathway in oridonin-induced reproductive toxicity[J]. Toxics, 2024, 12(5): 339.
[31] Lin T C, Germagian A, Liu Z H. The NF-κB signaling and Wnt/β- catenin signaling in MCF-7 breast cancer cells in response to bioactive components from Mushroom Antrodia camphorata[J]. Am J Chin Med, 2021, 49(1): 199-215.
[32] Zhang X Y, Qin X Y, Shen H H, et al. IL-27 deficiency inhibits proliferation and invasion of trophoblasts via the SFRP2/Wnt/β- catenin pathway in fetal growth restriction[J]. Int J Med Sci, 2023, 20(3): 392-405.
[33] Zhao M Y, Xu P, Liu Z, et al. Retraction Note: dual roles of miR-374a by modulated c-Jun respectively targets CCND1-inducing PI3K/AKT signal and PTEN-suppressing Wnt/β-catenin signaling in non-small-cell lung cancer[J]. Cell Death Dis, 2021, 12(7): 681.
Options
Outlines

/

〈 〉