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

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 of ZNF668 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.