Hepatocellular carcinoma-derived lactate-mediated histone H3K18 lactylation modifies S100A2 transcription to promote proliferation, migration, invasion and epithelial-mesenchymal transition of hepatocellular carcinoma cells

doi:10.12122/j.issn.1673-4254.2026.09.06

Abstract

Abstract:

Objective To investigate the role of histone H3 lysine 18 lactylation (H3K18la) regulating the transcription of S100 calcium-binding protein A2 (S100A2) in the development of hepatocellular carcinoma (HCC) and its molecular mechanism. Methods HCC single-cell transcriptome sequencing data were used to screen the differentially expressed genes and pathways. Intracellular lactate and histone lactylation were measured in HCC and normal hepatocyte cell lines. The effects of glycolysis inhibitors (2-DG/Oxamate) on proliferation, migration, and invasion of the cells were evaluated, and ChIP-qPCR was used to detect the enrichment of H3K18la at the S100A2 promoter. TCGA database was used to analyze the association of S100A2 expression with the prognosis of HCC patients. S100A2 overexpression and knockdown cell models were established, and rescue experiments using 2-DG or lactate were performed to validate the role of H3K18la in mediating the effects of S100A2. The interaction between S100A2 and KRT6A proteins was validated by STRING, molecular docking, and Co-IP. Western blotting was performed to determine the nuclear expression of β‑catenin and the EMT markers (vimentin and E‑cadherin) in HCC cells, and to elucidate the regulatory effect of H3K18la on the Wnt/β‑catenin pathway. Results Glycolysis and pyruvate metabolism pathways were upregulated in HCC, and lactate levels was elevated in HCC cells. Pan-Kla and H3K18la were significantly overexpressed in HCC cell lines, especially under hypoxia. Glycolysis inhibitors dose-dependently reduced H3K18la and suppressed malignant phenotypes of HCC cells. S100A2 mRNA and protein levels were upregulated in HCC, and its high expression was correlated with poor prognosis. S100A2 promoted HCC cell proliferation, migration, and invasion, which were reversed by 2‑DG. S100A2 directly interacted with KRT6A, the expression of nuclear β‑catenin was reduced after KRT6A knockdown and increased after KRT6A overexpression; exogenous lactate reversed KRT6A overexpression‑induced inhibition of β‑catenin, which was blocked by XAV939. Treatment with 2‑DG lowered the expressions of β‑catenin and EMT markers, which were reversed by S100A2 or KRT6A overexpression. Conclusion HCC-derived lactate enhances H3K18la modification, which activates S100A2 transcription to positively regulate the Wnt/β‑catenin pathway, ultimately promoting the proliferation, migration, invasion and EMT of HCC cells.

Key words: histone lactylation, S100A2, hepatocellular carcinoma, Wnt/β?catenin signaling pathway, epithelial-mesenchymal transition

Liping HUANG, Yu SHAO, Wanbing ZHOU, Jingping HE. Hepatocellular carcinoma-derived lactate-mediated histone H3K18 lactylation modifies S100A2 transcription to promote proliferation, migration, invasion and epithelial-mesenchymal transition of hepatocellular carcinoma cells[J]. Journal of Southern Medical University, 2026, 46(9): 2056-2068.

Figures/Tables 11

Tab.1 Targeted sequences of siRNA

Tab.2 Primers for RT-qPCR

Tab 3 Primers for ChIP-qPCR

Fig.1 Changes in pyruvate metabolic process and lactate levels in HCC. A-C: ScRNA-seq analysis of HCC tissues and paired adjacent normal liver tissues from 3 patients with primary HCC. D: Scatter plot of DEGs between normal and tumor samples. Red dots, upregulated genes in tumors; blue dots, downregulated genes; gray dots, genes without significant changes. Selection criteria: adjusted P value (Padj)<0.05 and |log2(fold change)|≥1. E: Schematic illustration of the glycolytic pathway. F: GSEA showing enrichment of pyruvate metabolism in HCC. G: GO enrichment analysis of upregulated DEGs in epithelial cells from tumor samples. H: Lactate levels in HCC and normal hepatic cell lines. Data are presented as Mean±SD (n=3). **P<0.01 vs L-O2 group.

Fig.2 Glycolysis-driven H3K18la promotes HCC progression. A: Western blotting of Pan Kla and H3K18la levels in HCC and normal hepatic cell lines. B, C: Western blotting of H3K9la, H3K18la, and H4K8la levels in HepG2 cells under normoxia and hypoxia. D: Schematic of glycolytic pathway and its inhibitors. E, F: Western blotting and of H3K18la levels following treatment with graded concentrations of 2-DG and oxamate for 24 h in hypoxia (1% oxygen). G: CCK-8 assay for the effect of 2-DG and oxamate for 24 h in hypoxia (1% oxygen) on the proliferation of HepG2 cells. H-I: Colony formation assay of HepG2 cells in different treatment groups with hematoxylin staining. J-K: Transwell migration and invasion assays of HepG2 cells in different treatment groups with crystal violet staining (Scale bar=100 μm). Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs Normal, Normoxia or Ctrl group; ^P<0.05, ^^^P<0.001 vs 2-DG (1 mmol/L) or oxamate (1 mmol/L) group; &&P<0.01 vs 2-DG (2 mmol/L) or oxamate (2 mmol/L) group.

Fig.3 H3K18la-mediated transcriptional activation of S100A2 is associated with poor prognosis in HCC. A: ChIP-qPCR analysis of H3K18la enrichment at the S100A2 promoter. B, C: Western blotting of S100A2 expression in HepG2 cells under hypoxia at indicated time points. D, E: Western blotting of S100A2 expression in HepG2 cells treated with lactate at indicated time points. F-H: Western blotting of H3K18la and S100A2 expression in HepG2 cells treated with 2-DG, lactate or 2-DG with lactate. I: Analysis of S100A2 expression in LIHC using TCGA database. J: Kaplan-Meier analysis of the association between S100A2 expression and prognosis in LIHC patients. K, L: Western blotting of S100A2 expression in HCC and normal hepatic cell lines. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs IgG, Hypoxia (0 h), Lactate (0 mmol/L), Ctrl or L-O2 group; ^^P<0.01, ^^^P<0.001 vs Hypoxia (6 h), Lactate (5 mmol/L) group or Lactate group; &&P<0.01, &&&P<0.001 vs Hypoxia (12 h) or Lactate (10 mmol/L) group.

Fig.4 S100A2 promotes proliferation, migration, and invasion of HCC cells. A-C: RT-qPCR and Western blotting of S100A2 mRNA and protein expression in 5 HCC cell lines. D-G: Western blotting and RT-qPCR analysis of S100A2 expression in HCC cells following overexpression or knockdown. H-I: CCK-8 assay for cell proliferation in different treatment groups. J, K: Colony formation assay of the cells with hematoxylin staining. L, M: Transwell migration and invasion assays with crystal violet staining (Scale bar=100 μm). Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs si-Ctrl or Ctrl group.

Fig.5 H3K18la modification promotes proliferation, migration, and invasion of HCC cells by regulating S100A2 expression. A: Western blotting of H3K18la and S100A2 expression in HepG2 cells in different treatment groups. B, C: Colony formation assay with hematoxylin staining. D: Transwell migration and invasion assays with crystal violet staining(Scale bar=100 μm). E: Western blotting of H3K18la and S100A2 expression in Huh-7 cells in different treatment groups. F, G: Colony formation assay with hematoxylin staining. H: Transwell migration and invasion assays with crystal violet staining (Scale bar=100 μm). Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs si-Ctrl or Ctrl group; ^^P<0.01, ^^^P<0.001 vs si-S100A2 or S100A2 group.

Fig.6 Protein-protein interaction between S100A2 and KRT6A. A: Protein-protein interaction network of S100A2. B: Surface representation of protein docking model between S100A2 (pink) and KRT6A (blue) with docking residues. C, D: Co-IP assay for detecting the interaction between S100A2 and KRT6A.

Fig.7 KRT6A promotes HCC progression by regulating the Wnt/β-catenin signaling pathway and EMT. A,B: Western blotting of nuclear β‑catenin protein levels following KRT6A knockdown and overexpression; C: Western blotting of nuclear β-catenin in different treatment groups. D: Western blotting analysis of nuclear β-catenin in different treatment groups. E: Western blotting of vimentin and E-cadherin expression in different treatment groups. F-G: Western blotting analysis of Vimentin and E-cadherin expression following KRT6A knockdown and overexpression. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs si-Ctrl or Ctrl group; ^^^P<0.001 vs Lactate or 2-DG group.

Fig.8 Mechanism of H3K18la for activating the S100A2/KRT6A/β-catenin signaling axis to promote the EMT process in HCC.

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