H4K5 lactylation - ENO2 loop drives glycolysis and HCC progression.

H4K5 lactylation - ENO2 loop drives glycolysis and HCC progression - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice JHEP Rep . 2026 Feb 17;8(5):101786. doi: 10.1016/j.jhepr.2026.101786 Search in PMC Search in PubMed View in NLM Catalog Add to search H4K5 lactylation - ENO2 loop drives glycolysis and HCC progression Masahiro Yamane Masahiro Yamane 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan 2 Department of Hepatobiliary and Pancreatic Surgery, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan Find articles by Masahiro Yamane 1, 2 , Yoshimitsu Akiyama Yoshimitsu Akiyama 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan Find articles by Yoshimitsu Akiyama 1, ⁎ , Shu Shimada Shu Shimada 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan Find articles by Shu Shimada 1 , Shuichi Watanabe Shuichi Watanabe 2 Department of Hepatobiliary and Pancreatic Surgery, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan Find articles by Shuichi Watanabe 2 , Megumi Hatano Megumi Hatano 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan Find articles by Megumi Hatano 1 , Shu Tsukihara Shu Tsukihara 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan 3 Department of Surgery, The Jikei University School of Medicine, Tokyo, Japan Find articles by Shu Tsukihara 1, 3 , Suguru Miyazawa Suguru Miyazawa 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan 2 Department of Hepatobiliary and Pancreatic Surgery, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan Find articles by Suguru Miyazawa 1, 2 , Hanako Tamura Hanako Tamura 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan 2 Department of Hepatobiliary and Pancreatic Surgery, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan Find articles by Hanako Tamura 1, 2 , Atsushi Nara Atsushi Nara 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan 2 Department of Hepatobiliary and Pancreatic Surgery, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan Find articles by Atsushi Nara 1, 2 , Keita Kodera Keita Kodera 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan 3 Department of Surgery, The Jikei University School of Medicine, Tokyo, Japan Find articles by Keita Kodera 1, 3 , Kohei Okazaki Kohei Okazaki 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan 3 Department of Surgery, The Jikei University School of Medicine, Tokyo, Japan Find articles by Kohei Okazaki 1, 3 , Yoshiaki Tanji Yoshiaki Tanji 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan 3 Department of Surgery, The Jikei University School of Medicine, Tokyo, Japan Find articles by Yoshiaki Tanji 1, 3 , Yosuke Igarashi Yosuke Igarashi 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan 3 Department of Surgery, The Jikei University School of Medicine, Tokyo, Japan Find articles by Yosuke Igarashi 1, 3 , Atsushi Kamachi Atsushi Kamachi 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan 4 Division of Gastroenterological, Hepato-Biliary-Pancreatic, Transplantation and Pediatric Surgery, Department of Surgery, Shinshu University School of Medicine, Matsumoto, Japan Find articles by Atsushi Kamachi 1, 4 , Kentaro Umemura Kentaro Umemura 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan 4 Division of Gastroenterological, Hepato-Biliary-Pancreatic, Transplantation and Pediatric Surgery, Department of Surgery, Shinshu University School of Medicine, Matsumoto, Japan Find articles by Kentaro Umemura 1, 4 , Koya Yasukawa Koya Yasukawa 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan 4 Division of Gastroenterological, Hepato-Biliary-Pancreatic, Transplantation and Pediatric Surgery, Department of Surgery, Shinshu University School of Medicine, Matsumoto, Japan Find articles by Koya Yasukawa 1, 4 , Keiichi Akahoshi Keiichi Akahoshi 2 Department of Hepatobiliary and Pancreatic Surgery, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan Find articles by Keiichi Akahoshi 2 , Kenichi Ohashi Kenichi Ohashi 5 Department of Human Pathology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan Find articles by Kenichi Ohashi 5 , Daisuke Ban Daisuke Ban 2 Department of Hepatobiliary and Pancreatic Surgery, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan Find articles by Daisuke Ban 2 , Shinji Tanaka Shinji Tanaka 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan Find articles by Shinji Tanaka 1, ⁎ Author information Article notes Copyright and License information 1 Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan 2 Department of Hepatobiliary and Pancreatic Surgery, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan 3 Department of Surgery, The Jikei University School of Medicine, Tokyo, Japan 4 Division of Gastroenterological, Hepato-Biliary-Pancreatic, Transplantation and Pediatric Surgery, Department of Surgery, Shinshu University School of Medicine, Matsumoto, Japan 5 Department of Human Pathology, Graduate School of Medicine, Institute of Science Tokyo, Tokyo, Japan ⁎ Corresponding authors. Address: Department of Molecular Oncology, Graduate School of Medicine, Institute of Science Tokyo, 1-5-45 Yushima, Bunkyo-Ku, Tokyo 113-8519, Japan. Tel.: +81-3-5803-5186 (Y. Akiyama) or +81-3-5803-5182 (S. Tanaka); fax: +81-3-5803-0125. yakiyama.monc@tmd.ac.jp tanaka.monc@tmd.ac.jp Received 2025 Aug 11; Revised 2026 Feb 7; Accepted 2026 Feb 11; Collection date 2026 May. © 2026 The Author(s) This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). PMC Copyright notice PMCID: PMC13091297  PMID: 41962496 Abstract Background & Aims Histone lactylation is a lactate-dependent epigenetic modification driven by glycolysis. Although elevated histone lactylation occurs in various cancers, the biological relevance of histone H4 lactylation in hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to investigate the clinicopathological relevance of histone H4 lysine 5 lactylation (H4K5la) and its role in regulating lactate metabolism to promote HCC progression. Methods H4K5la, H3K18la, and panKla (global lactylated lysine) expression levels were evaluated by immunohistochemistry in HCC tissues (n = 104). RNA sequencing and chromatin immunoprecipitation qPCR analyses were performed in HCC cells treated with lactate to identify downstream target genes regulated by H4K5la. Functional validation of these targets was conducted using knockdown and overexpression experiments, and biological effects were assessed in vitro and in vivo . Results Elevated H4K5la, H3K18la, and panKla levels were associated with poor prognosis, and H4K5la was identified as an independent prognostic factor of overall survival ( p = 0.0064). Upon lactate treatment, four genes including the glycolytic enzyme enolase 2 ( ENO2) were identified in three HCC cell lines, showing enrichment of H4K5la in their promoter regions. ENO2 knockdown reduced intracellular lactate and H4K5la levels, and in vivo tumorigenicity in HCC cells ( p <0.001), whereas ENO2 overexpression exerted the opposite effects. These findings indicated a positive feedback loop: H4K5la increased ENO2 expression, which in turn amplified glycolysis and lactate production, further elevating H4K5la levels. Moreover, ENO2 expression closely correlated with H4K5la levels in HCC tissues. Conclusions High histone H4K5la serves as an independent prognostic biomarker in HCC and promotes tumor progression via a positive feedback loop with ENO2. Targeting this H4K5la-ENO2 axis may represent a novel therapeutic strategy for patients with HCC and the hyper-lactylation phenotype. Impact and implications Despite limited understanding of the role of histone H4 lactylation in cancer, this study identifies H4K5la as a critical epigenetic regulator of hepatocellular carcinoma (HCC) progression. Lactate-induced elevation of H4K5la enhances ENO2 expression, which in turn increases H4K5la levels, establishing a positive H4K5la–ENO2 feedback loop in HCC. These data highlight that elevated H4K5la and ENO2 expression are potential prognostic biomarkers and therapeutic targets in patients with HCC. Keywords: histone lactylation, H4K5, glycolysis, positive feedback loop, lactate metabolism Graphical abstract Open in a new tab Highlights • Elevated H4K5la levels are associated with poor prognosis in patients with HCC. • H4K5la promotes HCC progression by epigenetically activating tumor-promoting genes. • A positive feedback loop of ENO2-H4K5la contributes to HCC progression. • The H4K5la-ENO2 pathway is a promising therapeutic target for HCC. Introduction Hepatocellular carcinoma (HCC) remains a significant global health challenge, ranking as the fourth leading cause of cancer-related mortality worldwide. 1 In 2020, approximately 1,000,000 new cases were diagnosed, resulting in an estimated 650,000 deaths. 2 The aggressive nature of HCC, often diagnosed at advanced stages, coupled with the limited effectiveness of current therapeutic options, contributes to its poor prognosis. 3 Despite advances in curative treatments, such as surgical resection, liver transplantation, and targeted molecular therapies, overall survival rates remain unsatisfactory, 4 highlighting the urgent need for innovative therapeutic strategies to improve patient outcomes. Recent machine learning–based models underscore the heterogeneity of HCC and the need for robust molecular biomarkers to improve patient stratification. 5 Metabolic reprogramming in cancer, particularly the activation of glycolysis under aerobic conditions (known as the Warburg effect), has emerged as a promising therapeutic target. [6] , [7] , [8] Glycolysis is a multifaceted metabolic pathway that plays an essential role in cancer cell survival and proliferation. Moreover, the metabolic shift in glycolysis has been shown to result in increased lactate production, which in turn acidifies the tumor microenvironment. [8] , [9] , [10] Enhanced glycolysis and subsequent lactate accumulation are closely involved in intracellular signaling and gene expression regulation. 6 , 11 Lactate inhibits histone deacetylase (HDAC) activity, thereby altering gene expression and highlighting a mechanistic link between glycolysis and epigenetic modifications in colorectal cancer cells. 12 Histone lactylation is a recently discovered post-translational modification that plays an essential role in regulating gene transcription. [13] , [14] , [15] , [16] This modification is catalyzed by histone acetyltransferases (HATs) and removed by HDACs, which are referred to as writer and eraser proteins, respectively. 16 Lactylation has been observed at multiple lysine residues on core histones, including histone H3 ( e.g . H3K9 and H3K18) and H4 ( e.g. H4K5 and H4K16), across a range of physiological and pathological conditions such as cancer, inflammation, embryogenesis, and neurodegeneration. [13] , [14] , [15] , 17 Among the lysine lactylation sites identified, 13 H3K18la is recognized as a major epigenetic mark in cancer, including HCC, with elevated levels strongly associated with malignant progression and poor prognosis. 17 , 18 Moreover, H3K18la-mediated transcriptional activation of glycolysis- and tumor-promoting genes has been reported in multiple types of cancer. 19 Increased H3K9la and H3K56la levels have been correlated with poor clinical outcomes in HCC. 20 Histone H3 lactylation has been extensively studied across various cancer types and the positive feedback loop between glycolysis and H3 lactylation has been characterized in tumor metabolism. 19 , 21 Recently, high H4K5la and H4K56la expression has been reported in several cancers, including breast cancer and neuroblastoma. 15 , 22 , 23 However, compared with the role of H3 lactylation in cancers, the clinicopathological relevance, downstream target genes, and tumor metabolism of histone H4 lactylation remain largely unexplored, particularly in HCC. In this study, we demonstrate that elevated H4K5la is an independent prognostic factor in patients with HCC. We further identify ENO2 , a glycolysis-related gene, as a direct target gene of H4K5la and uncover a positive ENO2–H4K5la feedback loop that increases glycolysis and promotes tumor progression. Our findings highlight H4K5la as a potential therapeutic target for HCC. Materials and methods Ethics statement All experimental protocols were approved by the Institutional Review Board (permission number G2017-018, Medical Research Ethics Committee for Life Science, Institute of Science Tokyo; G2018-132C10, Medical Research Ethics Committee for Genetic Research, Institute of Science Tokyo). All animal experiments were performed in compliance with the Guidelines for the Proper Conduct of Animal Experiments issued by the Science Council of Japan (permission number A2021-059C2, Institutional Animal Care and Use Committee, Institute of Science Tokyo). Human HCC tissues This study included 104 patients who underwent hepatectomy for HCC at the Institute of Science Tokyo Hospital between 2013 and 2016. Before surgery, written informed consent was obtained from all participants, and patient data were anonymized in accordance with the ethical guidelines of the Declaration of Helsinki. Descriptive data were collected by reviewing patients’ medical records. Human HCC cell lines Three human HCC cell lines, HLF (RRID: CVCL_2947), PLC/PRF/5 (RRID: CVCL_0485), and SNU475 (RRID: CVCL_0497), were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan) and the American Type Culture Collection (Manassas, VA, USA). The authentication of these cell lines was confirmed by short tandem repeat DNA fingerprinting (BEX, Tokyo, Japan). Detailed information on the cell culture conditions is provided in the supplementary information. All cells were routinely tested for mycoplasma contamination using the e-Myco plus Mycoplasma PCR Detection Kit (Cat# 25237, iNtRON Biotechnology, Seongnam, Korea) and were confirmed to be mycoplasma-free. Cells were cultured in medium containing 20 mM lactate (Wako, Osaka, Japan) for 24 h to induce histone H4 lactylation. This concentration was selected based on previous in vitro studies of histone lactylation [11] , [12] , [13] and was within the reported range of lactate levels in solid tumors (10–40 mM). 24 Immunohistochemistry (IHC) Human HCC tissue samples were sectioned at 4 μm, and all IHC was performed using a Histofine Simple Stain MAX PO® system (Cat# 424152, Nichirei Biosciences, Tokyo, Japan) according to the manufacturer’s instructions. The primary antibodies against H3K18la, H4K5la, H4K5ac, panKla (global lactylated lysine), and ENO2 used in this study are summarized in Table S1 . Immunoreactivity for histone lactylation or acetylation was evaluated via a blinded assessment of the staining score based on both the proportion of stained cells and the staining intensity. The proportion of stained tumor cells was scored as follows: 0 (0–10%), 1 (11–30%), 2 (31–50%), 3 (51–70%); and 4 (71–100%). Staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The total score (TS) was calculated by multiplying the intensity and proportion scores; TS >7 indicates strong expression. Immunoreactivity for ENO2 expression was based on the staining intensity in the cytoplasm relative to adjacent non-tumor liver tissues, as previously described. 25 Samples were classified as strong if the cytoplasmic staining intensity in the target tissue was markedly higher than that in adjacent non-tumor liver tissues, whereas weak expression was defined as a cytoplasmic staining intensity that was only slightly above background levels and comparable to that of adjacent non-tumor liver tissues. Functional analyses of histone lactylation Detailed experimental procedures for RNA and protein analyses, histone lactylation assays, cell proliferation, and the measurement of intracellular metabolites are shown in the supplementary information. The antibodies and PCR primer sets used are listed in Tables S1–S3 , and the small-interfering RNA and short-hairpin RNA sequences used in this study are listed in Table S4 . Animal experiments Tumorigenicity was assessed in a xenograft model of PLC/PRF/5 cells. Immunodeficient nude mice (KSN/Slc, male, 4 weeks old) were purchased from Sankyo Labo Service, Japan. All mouse procedures were approved by the Institutional Animal Care and Use Committee of the Institute of Science Tokyo, conducted according to its guidelines, and adhered to the ARRIVE guidelines for reporting animal research. The detailed animal experimental procedures, including animal numbers, randomization, and blinding, are provided in the supplementary information. For the xenograft tumor model, 1 × 10 6 PLC/PRF/5 cells with ENO2 suppression and re-expression of ENO2 were suspended in 100 μl of Matrigel (BD Biosciences) and then subcutaneously injected into two sites per mouse (bilateral flanks) in KSN/Slc mice (n = 6). For pharmacological inhibition (n = 6), the ENO2 inhibitor POMHEX (Selleck Chemicals, Houston, TX, USA; Cat. No. S9830) was administered via intraperitoneal (i.p.) injection at 40 mg/kg every 2 days after tumors reached 100–150 mm 3 . The tumor volume (V) was calculated as V = (Length × Width 2 )/2. Mice were euthanized upon reaching predefined humane endpoints ( e.g . tumor volume >2,000 mm 3 ). Statistical analysis All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) or GraphPad Prism 10.3.0 (GraphPad Software, La Jolla, CA, USA). A two-sided Student’s t test was used to analyze differences in continuous values between the two independent groups. Spearman’s rank correlation coefficient is denoted as ρ. The χ 2 test or Fisher’s exact test was applied to analyze categorical variables. Survival curves were constructed using the Kaplan–Meier method and compared using the log-rank test. Significant variables were subjected to multivariate analysis using the Cox proportional hazards model after the univariate analysis. n.s. indicates no significant difference at p <0.05. Results Elevated levels of H4K5la are associated with poorer prognosis in patients with HCC Given the clinical importance of lactylation in HCC progression, we investigated the clinicopathological relevance and prognostic implications of H4K5la, H3K18la, and panKla expression in the HCC tissue samples ( Fig. S1A ). IHC analysis demonstrated that H4K5la expression was markedly higher in HCC tissues than in adjacent non-tumor liver tissues ( Fig. 1 A,B). H3K18la levels were also high in HCC tissues; however, several cases showed equally high levels in adjacent non-tumor liver tissues ( Fig. 1 B). High H4K5la expression (51.0%, 53/104) was significantly associated with aggressive clinicopathological features, including larger tumor size and poorer differentiation, and was predictive of worse overall survival (OS, p < 0.0001) and disease-free survival (DFS, p = 0.0009) ( Table 1 , Fig. 1 C). Although neither H3K18la (56.9%, 58/102) nor panKla (49%, 49/100) exhibited notable clinicopathological associations compared with H4K5la, both were still indicative of poor prognosis ( Tables S5 and S6 , Fig. 1 C), consistent with previous findings on their prognostic relevance in HCC. 21 Univariate analysis revealed that high H4K5la, H3K18la, and panKla expression levels were significantly associated with OS and DFS ( Table S7 ). However, multivariate analysis identified high H4K5la expression as the only independent prognostic factor for OS ( p < 0.0064, Fig. 1 D). To further investigate the importance of histone modification at the H4K5 site, the levels of histone acetylation (H4K5ac) in HCC tissues were assessed. Although high H4K5ac expression was observed in HCC (55.1%, 54/98), it was not significantly correlated with tumor malignancy or patient prognosis ( Table S8 , Fig. S1B ). Subgroup analysis stratified by pathological stage also revealed that high H4K5la expression was significantly associated with poor OS in both early-stage (pStage I/II) and advanced-stage (pStage III/IV) diseases ( Fig. S2 ). These data suggest that H4K5la serves as a valuable biomarker for refining risk stratification within the existing clinical staging system and that high H4K5la is more specifically associated with HCC progression. Fig. 1. Open in a new tab Clinical significance of histone lactylation in HCC. (A) Representative IHC staining of H4K5la and H&E staining of a human HCC tissue section containing both tumor and adjacent non-tumor liver tissue. The lower panels show a higher magnification of the regions indicated by dotted boxes in the upper panel. The scale bars represent 100 and 250 μm. (B) Violin plot comparing H4K5la (n = 99) and H3K18la (n = 100) staining score between tumor and non-tumor liver tissues in human HCC samples. H4K5la expression was markedly higher in HCC tissues compared with adjacent non-tumor liver tissues. Statistical significance was determined using the Mann-Whitney U test. Asterisks indicate statistical significance (∗ p < 0.05, ∗∗∗ p < 0.001). (C) Kaplan–Meier curves for OS and DFS of 104 patients with HCC grouped by panKla, H4K5la, and H3K18la expression levels. Strong panKla, H3K18la, and H4K5la expression levels were significantly associated with poorer OS and DFS. p -values were calculated using the log-rank test. (D) Multivariate analysis of OS and DFS in patients with HCC based on clinicopathological characteristics and panKla, H4K5la, and H3K18la expression. DFS, disease-free survival; H3K18la, histone H3 lysine 18 lactylation; H4K5la, histone H4 lysine 5 lactylation; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; OS, overall survival; panKla, global lactylated lysine. Table 1. Relationship between H4K5la expression and clinicopathological factors. Variable H4K5la low (n = 51) H4K5la high (n = 53) p value Patient characteristics Age (years) 69.3 ± 10.1 71.9 ± 7.5 0.16 Sex (male/female) 40/11 42/11 1 Body mass index (kg/m 2 ) 24.0 ± 4.5 24.1 ± 3.5 0.90 Liver status Viral infection 27 (52.9%) 32 (60.4%) 0.69 FIB-4 index 3,83 ± 3.31 3.76 ± 2.40 0.89 ALBI score -2.62 ± 0.33 -2.61 ± 0.40 0.79 Tumor Tumor size, >50 mm 13 (25.5%) 24 (45.3%) 0.042∗ Tumor number, St vs. Mt 42/9 41/12 0.36 Differentiation, well vs . mod/poor 26/25 9/44 <0.001∗ Portal vein invasion, Vp0 vs . Vp1-4 36/15 33/20 0.60 Hepatic vein invasion, Vv0 vs . Vv1-3 45/6 44/9 0.46 Hepatic artery invasion, Va0 vs . Va1-3 50/1 52/1 1.00 Bile duct invasion, Vb0 vs . Vb1-3 49/2 50/3 0.62 pStage, I,II vs. III, IV 37/14 36/17 0.67 Alpha-fetoprotein, >200 ng/ml 10 (19.6%) 16 (30.2%) 0.26 PIVKA-II, >100 mAU/ml 18 (35.3%) 34 (64.2%) 0.0058∗ Open in a new tab Values are presented as mean ± SD or n (%) as appropriate. A two-sided Student’s t test or Mann–Whitney U test was applied to assess differences in continuous data between two independent groups. A chi-square test was applied to assess differences in two categorical variables between two independent groups. A value of p <0.05 was considered statistically significant (∗). ALBI, albumin-bilirubin; FIB-4, fibrosis-4; mod, moderately differentiated; Mt, multiple tumor; poor, poorly differentiated; St, solitary tumor; well, well-differentiated. Effects of environmental changes on histone H4K5la We previously reported that H3K18la levels are elevated in gastric cancer cells under high-lactate conditions but are diminished when grown in glucose-free media or treated with glycolytic inhibitors. 21 Based on these findings, we investigated the effects of environmental changes on histone lactylation in three HCC cell lines (HLF, PLC/PRF/5, and SNU475). H3K18la levels decreased under glucose deprivation and increased under lactate treatment (20 mM). H4K5la levels also decreased under glucose deprivation, whereas high lactate levels markedly increased its levels ( Fig. 2 A). Peptide competition for H4K5la completely abolished the H4K5la signal, indicating specificity of H4K5la detection in these cell lines ( Fig. S3 ). Treatment with the glycolytic inhibitors, 2-deoxyglucose and oxamate, significantly reduced the levels of H4K5la and H3K18la ( Fig. 2 B), indicating that these histone lactylation markers are linked to glycolysis in HCC. Fig. 2. Open in a new tab Effects of environmental changes on histone lactylation in HCC. (A) Western blot analysis of H4K5la and H3K18la levels in HCC cell lines treated with high lactate concentrations (20 mM) and grown under glucose deprivation for 24 h panH4 was used as an internal control. (B) Effects of glycolytic inhibitor treatment with oxamate and 2-DG on H4K5la and H3K18la expression in HCC cells. (C) Experimental workflow employed to identify and validate genes associated with H4K5la and HCC malignancy. (D) Venn diagram based on RNA-seq data representing 13 commonly upregulated genes across all three cell lines with lactate treatment, as shown by overlaps. (E-F) Significantly upregulated pathways detected by GSEA for three HCC cell lines with lactate treatment. Normalized enrichment scores for key hallmark pathways, including hypoxia and glycolysis, in HLF, PLC/PRF/5, and SNU475 cells (E) and GSEA plots demonstrating the significant activation of the glycolysis pathway (F). 2-DG, 2-deoxyglucose; GSEA, gene set-enrichment analysis; H3K18la, histone H3 lysine 18 lactylation; H4K5la, histone H4 lysine 5 lactylation; HCC, hepatocellular carcinoma; panH4, total histone H4; RNA-seq, RNA sequencing. Because histone lactylation is strongly associated with transcriptional activation, 12 , 14 , 16 , 18 , 19 , 21 , 23 we investigated the H4K5la-dependent downstream target genes that were upregulated by lactate treatment in HCC cells. The experimental workflow is shown in Fig. 2 C. RNA-sequencing analysis identified 13 genes that were commonly upregulated in all three cell lines after 24 h of lactate treatment (20 mM) compared to untreated controls (log 2 fold change >0.5, p value <0.05; Figs. 2 D, S4A ). Gene set-enrichment analysis identified several key pathways associated with malignant progression, including glycolysis, hypoxia, and apical junction ( Fig. 2 E,F). Identification of downstream target genes of H4K5la in HCC Among the 13 genes detected by RNA-sequencing analysis ( Fig. 2 C), the expression of 12 genes, except for FAM114A1 , was upregulated in all three cell lines treated with lactate ( Fig. 3 A and S4B ). To identify the specific downstream targets of H4K5la in HCC, we assessed the enrichment of histone modifications (H4K5la, H4K5ac, and H3K18la) in PLC/PRF/5 and HLF cells using chromatin immunoprecipitation (ChIP)-qPCR. Lactate treatment commonly increased the enrichment in H4K5la at the promoter regions of nine genes in both PLC/PRF/5 and HLF cells, five of which ( LMTK3, ENO2, ALDOC, TBC1D8B, and KANK3 ) showed no significant increase in H3K18la or H4K5ac levels at their promoter regions ( Fig. 3 B). Fig. 3. Open in a new tab Identification of target genes of H4K5la by RNA-seq and ChIP-seq. (A) Validation of RNA-seq results by qRT-PCR. qRT-PCR analysis confirmed significant upregulation of 12 out of the 13 candidate genes, excluding FAM114A1 , upon lactate treatment across all three HCC cell lines. The stability of 18S rRNA as a reference gene was validated by comparing its performance with that of other housekeeping genes, TBP and HPRT1 ( Fig. S4C ). Since 18S rRNA normalization most closely recapitulated the expression patterns observed in our RNA-seq dataset, compared to TBP and HPRT1 , we selected 18S rRNA as the internal control for qRT-PCR. (B) ChIP-qPCR at the promoter regions of 12 genes using antibodies against H4K5la (top), –H3K18la (middle), and H4K5ac (bottom). The aim of this study was to identify genes specifically upregulated through H4K5la. Four genes ( LMTK3 , ENO2 , ALDOC , and KANK3 ) were selected based on elevated H4K5la levels at their promoter regions, with no significant increases in H3K18la or H4K5ac. Gene desert regions were used as negative controls. Enrichments of H4K5la and H4K5ac at the gene deserts were normalized to total H4, whereas enrichment of H3K18la was normalized to total H3 to account for nucleosome occupancy. Gene desert regions showed no detectable amplification or only low H4K5la signals (Ct >36), which are displayed at the detection limit (★). (C) Representative ChIP-seq tracks showing H4K5la enrichment at the genomic regions of ENO2 and LMTK3 . (D) ChIP-qPCR analyses at the promoter and gene body regions of these two genes were performed to validate the ChIP-seq data. (E) Transcription factor binding motifs identified in the ENO2 promoter region. ChIP-seq and motif enrichment analysis (HOMER) identified binding sites for key transcription factors, including NFATC1, HOXC13, and GCM1, within the ENO2 promoter. The table presents the consensus sequences, genomic coordinates (hg38), and predicted binding sequences for each factor. Data are presented as the mean ± SD from three independent experiments, and statistical significance was determined using Student’s t test. Asterisks indicate statistically significant differences (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). ChIP, chromatin immunoprecipitation; H3K18la, histone H3 lysine 18 lactylation; H4K5ac, histone H4 lysine 5 acetylation; H4K5la, histone H4 lysine 5 lactylation; HCC, hepatocellular carcinoma; HOMER, hypergeometric optimization of motif enrichment; qRT-PCR, quantitative reverse transcription PCR. ChIP-seq and subsequent ChIP-qPCR analyses consistently validated a specific increase in H4K5la levels at the promoter regions but not in the gene body regions of four genes ( ENO2 , LMTK3, KANK3 , and ALDOC ) after lactate treatment ( Fig. 3 C,D, and S5A,B ). In contrast, ChIP-seq analysis revealed uniformly weak H4K5la signal intensity across the entire TBC1D8B locus, including the promoter and gene body regions, compared to the other four genes, suggesting that H4K5la plays a minimal role in the transcriptional regulation of TBC1D8B in PLC/PRF/5 cells ( Fig. S5C ). These data indicate that promoter-associated H4K5la is strongly correlated with the transcriptional activation of the four genes identified above. Therefore, we selected these genes for further studies. Accordingly, we performed motif analysis to search for specific sequences regulated by H4K5la and identified six motifs, including NFATC1 and HOXC13, in the ENO2 promoter region ( Fig. 3 E). EP300 acts as a histone lactylase in HCC As KAT5, KAT6B, CBP, and EP300 catalyze acetylation at multiple sites, including H4K5, 26 we examined whether these four HATs are responsible for H4K5la in HCC. Analysis of the TCGA database revealed that the expression levels of all four HATs in HCC tissues were higher than in non-tumor tissues ( Fig. 4 A), consistent with our findings of elevated H4K5la levels in HCC tissues. Remarkably, screening analysis using small-interfering RNA-mediated knockdown (KD) of these four HAT genes indicated a significant reduction in global H4K5la only upon EP300 -KD in HCC cells ( Fig. 4 B and S6 ). Fig. 4. Open in a new tab Functional analysis of EP300 as an H4K5la writer enzyme. (A) TCGA dataset analysis of mRNA expression levels of four HAT genes ( KAT5 , KAT6B , CREBBP , and EP300 ) in HCC. Red and black dots represent tumor and normal tissue samples, respectively. (B) Western blot analysis showing the effect of siRNA-mediated EP300 suppression (siEP300) on H4K5la levels in PLC/PRF/5 and HLF cells. Suppression of three additional HAT genes ( KAT5 , KAT6B , and CBP ) was also performed in HLF cells. (C) Effects of shRNA-mediated EP300 -KD in HCC cells. Stable cell lines were established using shEP300 #1, shEP300 #2, or negative control shRNA (NC) in two HCC cell lines. qRT-PCR analysis was performed to evaluate the KD efficiency of EP300. (D) Western blot analysis confirming the EP300 -KD and its effect on H4K5la levels in HCC cells. (E) qRT-PCR analysis showing the relative expression of four candidate genes in HCC cells with EP300 -KD in (shEP300 #1 and shEP300 #2) compared with shNC. (F) ChIP-qPCR analysis showing relative fold enrichment of H4K5la at the promoter regions of these four genes. Pan H4 and β-actin were used as internal controls for H4K5la and EP300 expression in Western blot analysis. 18S ribosomal RNA was used for qRT-PCR normalization. Relative levels of EP300 recruitment and H4K5la enrichment were normalized to Input DNA and pan H3, respectively, using ChIP-qPCR. Data are presented as the mean ± SD from three independent experiments, and statistical significance was determined using Student’s t test. Asterisks indicate statistical significance (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). ChIP, chromatin immunoprecipitation; H4K5la, histone H4 lysine 5 lactylation; HAT, histone acetyltransferase; HCC, hepatocellular carcinoma; KAT5, lysine acetyltransferase 5; KAT6B, lysine acetyltransferase 6B; NC, negative control; pan H4, total histone H4; qRT-PCR, quantitative reverse transcription PCR; shRNA, short hairpin RNA; siRNA, small interfering RNA; TCGA, The Cancer Genome Atlas. We generated HCC cell lines with stably suppressed EP300 expression ( EP300 -KD) and analyzed the effects of EP300 depletion on H4K5la and downstream gene expression. EP300 -KD significantly reduced global H4K5la levels and the expression of all four target genes ( Fig. 4 C-E). Furthermore, ChIP-qPCR analysis revealed decreased EP300 recruitment and H4K5la accumulation at the promoters of these genes in HCC cells with EP300 -KD ( Fig. 4 F). Knockdown of H4K5la-downstream target genes inhibits tumor malignancy The tumor-promoting functions of four H4K5la-downstream target genes have already been reported in various cancers. [27] , [28] , [29] , [30] , [31] , [32] To verify these functions in HCC cells, we performed small interfering RNA-mediated KD of these genes in HLF cells and evaluated their effects on cell proliferation, migration, and invasion. ENO2, LMTK3, and ALDOC, but not KANK3, significantly inhibited these oncogenic properties ( Fig. S7A and B ), indicating their involvement in promoting HCC progression. Importantly, only ENO2 -KD in HLF cells resulted in a significant reduction in H4K5la levels ( Fig. S7C ). Consistent with this finding, treatment with POMHEX, an ENO2 inhibitor, also reduced H4K5la levels after 24 h, whereas H3K18la levels remained unchanged ( Fig. S7D ). These results suggest a potential feedback regulatory mechanism in which ENO2 influences H4K5la levels in HCC cells. Positive feedback loop of glycolysis via H4K5la-ENO2 axis ENO2 is a critical enzyme in glycolysis. 30 To further investigate the mechanistic link between ENO2 and H4K5la in the glycolytic pathway, stable ENO2 -KD and ENO2 overexpression (OE) were established. ENO2 -KD resulted in a marked decrease in H4K5la levels, whereas ENO2 -OE led to an increase in H4K5la levels in both PLC/PRF/5 and HLF cells, compared to their respective controls ( Fig. 5 A,B). However, H4K5ac levels remained unchanged in HCC cells with ENO2 -KD or ENO2 -OE. ENO2 catalyzes the conversion of 2-phospho-D-glycerate to phosphoenolpyruvate (PEP) during glycolysis. Therefore, we evaluated the effects of ENO2 modulation on intracellular PEP, pyruvate, and lactate levels. As shown in Fig. 5 C,D, intracellular PEP, pyruvate, and lactate concentrations were significantly decreased in both PLC/PRF/5 and HLF cells with ENO2 -KD and increased in cells with ENO2 -OE. In addition, LDH enzymatic activity significantly decreased in ENO2 -KD cells and increased in ENO2 -OE cells, indicating that ENO2 promotes glycolysis and regulates H4K5la expression in HCC cells. Fig. 5. Open in a new tab Positive feedback loop of glycolysis and ENO2 promotes H4K5la. (A, B) Western blot analysis showing the effects of ENO2 -KD and ENO2 -OE in HCC cells. Stable HCC cells with ENO2 -KD and ENO2 -OE were established using shRNAs (shENO2 #1 or shENO2 #2) (A) and an overexpression vector (B), respectively. Negative control shRNA (shNC) and GFP-expressing (Control) HCC cells served as controls in each experiment. pan H4 and β-actin were used as internal controls for H4K5la and ENO2 expression. (C, D) Intracellular concentrations of the key glycolytic metabolites and LDH activity in cell lysates of ENO2 -KD and ENO2 -OE HCC cells, and LDH activities. PEP was measured using a fluorescence assay with a microplate reader (excitation: 550 nm, emission: 590 nm). Lactate, pyruvate, and LDH activity were measured in cell lysates by a colorimetric assay with absorbance at 450 nm. (E, F) Relative expression levels of H4K5la-regulated genes and H4K5la/H4K5ac enrichment levels of their promoter regions in HCC cells with ENO2 -KD (E) and ENO2 -OE (F). (G) Re-expression of ENO2 in PLC/PRF/5 cells with ENO2 -KD using shENO2 #1 targeting the ENO2 3’-UTR. ENO2 -OE rescued global H4K5la expression as well as the expression and H4K5la enrichments at the promoters of three target genes. (H) ENO2/glycolysis/H4K5la positive feedback loop. β-actin and pan H4 were used as internal controls for Western blot analysis. 18S ribosomal RNA was used for qRT-PCR normalization. Relative H4K5la and H4K5ac enrichment levels were normalized to pan H4 by ChIP-qPCR. Data are presented as the mean ± SD from three independent experiments, and statistical significance was determined using Student’s t test. Asterisks indicate statistically significant differences (∗∗ p < 0.01, ∗∗∗ p < 0.001). ChIP, chromatin immunoprecipitation; H4K5ac, histone H4 lysine 5 acetylation; H4K5la, histone H4 lysine 5 lactylation; HCC, hepatocellular carcinoma; KD, knockdown; OE, overexpression; PEP, phosphoenolpyruvate; qRT-PCR, quantitative reverse transcription PCR; shNC, negative control short hairpin RNA; shRNA, short hairpin RNA. ENO2 -KD resulted in decreased expression of H4K5la downstream target genes ( LMTK3, ALDOC, and KANK3 ) and decreased enrichment of H4K5la in their promoter regions in PLC/PRF/5 cells ( Fig. 5 E). Conversely, ENO2 -OE increased their expression and enhanced H4K5la enrichment in the promoters ( Fig. 5 F). No significant changes in H4K5ac enrichment at the promoter regions of these genes were observed upon ENO2- KD or ENO2 -OE, suggesting that ENO2 contributes to increasing H4K5la, but not H4K5ac. Because shENO2 #1 targets the 3’-untranslated region of ENO2 , we conducted ENO2 re-expression in PLC/PRF/5 cells with ENO2 -KD (shENO2 #1), as a rescue experiment. Re-expression of ENO2 rescued target gene expression and restored H4K5la enrichment at the promoters ( Fig. 5 G), functionally re-establishing the loop. These data demonstrate the existence of a positive feedback loop between H4K5la and ENO2 during glycolysis ( Fig. 5 H). Evaluation of the biological effects of ENO2-OE or -KD on PLC/PRF/5 cells We evaluated the role of ENO2 in tumorigenicity in vivo using immunodeficient nude mice ( Fig. S8A–H , n = 3). Subcutaneous xenograft tumors derived from ENO2 -KD PLC/PRF/5 cells grew more slowly than those in the control, with decreased H4K5la expression and fewer Ki67-positive cells. In contrast, ENO2 -OE xenografts grew faster, accompanied by increased H4K5la and Ki-67. H4K5ac levels did not change in these xenograft tumors. To further elucidate the role of ENO2 in the positive feedback loop of glycolysis, we performed in vivo ENO2 rescue experiments. Xenograft tumors derived from ENO2 -KD cells with ENO2 re-expression fully rescued the growth-inhibitory phenotype, restoring tumor volumes to levels comparable to those in the control group ( Fig. 6 A-C). Furthermore, tumors with ENO2 re-expression showed restored H4K5la levels and Ki67 positivity but not H4K5ac levels ( Fig. 6 D). Fig. 6. Open in a new tab Biological effects of ENO2 in vivo and clinical significance of ENO2 in HCC. (A–D) In vivo tumorigenicity of ENO2 -KD and its rescue models. A total of 1 × 10 6 PLC/PRF/5 cells with ENO2 -KD (shENO2 #1), ENO2 -KD with ENO2 re-expression (shENO2 #1 + ENO2 -OE), or negative control (shNC) were subcutaneously injected into two sites per mouse (bilateral flanks) in KSN/Slc nude mice (n = 6). Tumor growth was monitored for >26 days (A). Representative images of xenograft tumors (B) and final tumor volumes (C) are shown. D) IHC of ENO2, H4K5la, H4K5ac, and Ki67 in xenograft tumors. Re-expression of ENO2 restored the effects of ENO2 -KD. (E–H) In vivo therapeutic potential of the ENO2 inhibitor POMHEX. PLC/PRF/5 cells with ENO2 -OE were subcutaneously injected into KSN/Slc mice (n = 6, two injection sites per mouse), followed by treatment with POMHEX (40 mg/kg, i.p.) or vehicle (control) every 2 days (E: arrows indicate treatment). Final body weight (F), tumor volume (H), and photographs of xenograft tumors (G) are shown. (I) IHC of ENO2, H4K5la, H4K5ac, and Ki-67 in xenograft tumors treated with POMHEX. POMHEX treatment markedly suppresses tumor growth and reduces H4K5la and Ki67 levels in vivo . Scale bars: 250 μm. Student’s t test was used for tumor volumes (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; n.s., not significant). (J) Representative IHC staining for ENO2 in human HCC (strong vs. weak) and adjacent non-tumor liver tissues. Scale bars: 250 μm. (K) Kaplan–Meier curves for OS and DFS of 102 patients with HCC grouped by ENO2 expression levels. High ENO2 expression was significantly associated with poor OS and DFS, and p values were calculated using the log-rank test. DFS, disease-free survival; H4K5ac, histone H4 lysine 5 acetylation; H4K5la, histone H4 lysine 5 lactylation; HCC, hepatocellular carcinoma; i.p., intraperitoneal; IHC, immunohistochemistry; KD, knockdown; OE, overexpression; OS, overall survival; shNC, negative control short-hairpin RNA; shRNA, short-hairpin RNA; shENO2, short-hairpin RNA targeting ENO2. Next, we evaluated the therapeutic potential of the ENO2 inhibitor POMHEX. Xenograft tumors derived from ENO2 -OE cells exhibited accelerated growth compared with controls; however, treatment with POMHEX (40 mg/kg, i.p.) significantly suppressed ENO2-driven tumor progression ( Fig. 6 E-H). POMHEX treatment did not cause significant body weight loss, indicating minimal systemic toxicity ( Fig. 6 F). Furthermore, POMHEX treatment effectively decreased H4K5la and Ki67 levels in ENO2 -OE tumors ( Fig. 6 I). To assess the clinical relevance of ENO2 in HCC, we investigated the clinicopathological characteristics and prognostic significance of ENO2 expression in HCC tissue samples using IHC ( Fig. 6 J). The high-ENO2 expression group (48.0%, 49/102) exhibited a significantly larger tumor size, poorer differentiation, and a higher incidence of vascular invasion ( Table S9 ). Furthermore, high ENO2 expression was significantly associated with poor OS and DFS ( Fig. 6 K), consistent with previous findings based on TCGA-Liver Hepatocellular Carcinoma (LIHC) analysis. 29 , 30 It is worth noting that high ENO2 levels were strongly correlated with H4K5la ( p < 0.001), but not with panKla or H3K18la in HCC ( Table S9 ). Discussion Histone H3 lactylation has been extensively studied in cancer. [17] , [18] , [19] , [20] , [21] The findings of this study indicate that H4K5la expression is elevated in HCC and correlates with enhanced cell proliferation, migration, invasion, and tumorigenicity. Furthermore, we identify a novel positive feedback loop mediated by the H4K5la-ENO2 axis that links glycolytic metabolism to tumor progression. Elevated levels of panKla and histone lactylation have been increasingly associated with enhanced malignant progression and diminished patient prognosis across various tumors. [19] , [20] , [21] , [22] Our IHC results revealed that nuclear H4K5la expression was significantly enhanced in human HCC and correlated with disease advancement and unfavorable patient outcomes. Importantly, H4K5la was markedly elevated in HCC tissues but was minimal in adjacent non-tumor liver tissues, whereas H3K18la was partially elevated in adjacent non-tumor liver tissues, indicating that H4K5la is a more tumor-specific lactylation marker in HCC. Histone H4 lactylation occurs at multiple lysine residues, including K5, K8, K12, and K16. 33 However, studies on H4 lactylation in clinical specimens are limited due to the scarcity of site-specific, clinically reliable antibodies. 22 , 23 , 34 Our data strongly suggest that elevated H4K5la expression can serve as a crucial independent prognostic biomarker in HCC. Recent studies have identified numerous downstream targets of H3K18la and H3K9la in various cancer types, highlighting their roles in promoting oncogenic signaling and modulating immune responses. 35 Moreover, lactylation at the promoter and enhancer regions of H3K18 is critical for transcriptional activation. 36 , 37 In the present study, four genes ( LMTK3, ENO2, ALDOC, and KANK3 ) were specifically upregulated in an H4K5la-dependent manner. Furthermore, our data and previous reports highlighted the tumor-promoting functions of LMTK3, ENO2, and ALDOC ( Fig. S7B , [27] , [28] , [29] , [30] , [31] ). Although the previous studies have identified COL1A1 and PD-L1 as H4K5la target genes in acute myeloid leukemia and neuroblastoma, 15 , 24 the number of studies identifying H4K5la target genes in cancer remains limited. Our results indicated that H4K5la upregulated a diverse set of pro-oncogenic genes, thereby contributing to enhanced proliferation and malignant characteristics in HCC. Recent studies have highlighted that tumor metabolic reprogramming influences tumor progression through multiple biological pathways. Protein lactate modifications exert diverse oncogenic functions in HCC. For instance, ABCF1-K430 lactylation promotes its nuclear translocation and subsequent transcriptional activation of the HIF-1 signaling pathway, 38 whereas ASH2L-K312 lactylation enhances angiogenic gene expression. 39 Moreover, tryptophan metabolism modulates FTH1-driven ferritinophagy and ferroptosis in HCC, contributing to tumor growth and poor prognosis. 40 These observations underscore the importance of metabolite-responsive mechanisms linking metabolic reprogramming to diverse cellular processes during tumor progression. Notably, EP300 and CBP are among the first enzymes with the capacity to mediate lysine lactylation, employing lactyl-CoA as a cofactor to transfer lactyl groups to histone tails. 11 , 41 , 42 Accumulating evidence indicates a central role for EP300 in establishing histone/non-histone lactylation marks in cancers, including HCC. 38 , 43 In this study, EP300 -KD significantly reduced global H4K5la levels and H4K5la-mediated target gene expression in HCC cells. EP300 -OE correlates with a malignant phenotype and poor prognosis in patients with HCC. 44 , 45 Although HDAC1-3 exhibit delactylase activity for H4K5la in vitro , 41 high expression of HDAC1-3 is also associated with poor prognosis in HCC, 46 , 47 suggesting that these eraser proteins may not contribute to H4K5la elevation in HCC. Therefore, our findings suggest that EP300 expression reflects the malignant progression and poor prognosis associated with H4K5la in HCC and that EP300 functions as a specific writer of H4K5la in HCC. One of the key findings of this study is the identification of a positive feedback loop in which H4K5la, induced by elevated lactate levels resulting from enhanced glycolysis, directly upregulates ENO2 . Consistent with this loop, the ENO2-H4K5la axis increased intracellular levels of PEP, pyruvate, and lactate, as well as LDHA activity. Moreover, high ENO2 expression increases glycolytic flux in HCC cells. 48 Our findings provide evidence for an additional epigenetic regulatory mechanism underlying the glycolysis-driven positive feedback loop mediated by histone H4 lactylation in HCC ( Fig. 5 H). Bioinformatics analysis using JASPAR software has recently revealed that the ENO2 promoter region contains multiple HIF-1α binding sites. 48 We identified six predicted transcription factor-binding motifs, including NFATC1 and HOXC13. Further studies are required to elucidate the precise roles of these transcription factors in regulating ENO2 in conjunction with H4K5la. Regarding therapeutic implications, ENO2 is highly expressed and exerts pro-oncogenic effects in numerous cancer types, including HCC. 31 , 32 , [48] , [49] , [50] In this study, ENO2 was upregulated in HCC; however, its expression in non-tumor liver tissues was relatively low, suggesting a potentially favorable therapeutic window. Our data also revealed that high ENO2 expression was associated with poorer prognosis in patients with HCC and positively correlated with H4K5la expression in primary HCC cases. Therefore, ENO2 and H4K5la may serve as prognostic biomarkers for HCC and help identify patient subgroups more likely to benefit from ENO2-targeted therapies. From a therapeutic perspective, ENO2-targeted therapies have been explored for several tumor types, including head and neck squamous cell carcinoma, colorectal cancer, and ENO1 -deficient/1p36-deleted brain tumors, 51 although their clinical application has not yet been achieved. In our study, POMHEX effectively suppressed HCC progression in a mouse model of ENO2 -OE without significant weight loss, supporting its therapeutic potential. However, systemic administration of ENO2 inhibitors, such as POMHEX, has several limitations, including potential toxicity from off-target inhibition of ENO1 in erythrocytes. 51 Additionally, our findings provide evidence that inhibition of H4K5la by targeting EP300 is a promising strategy for suppressing ENO2 expression in HCC. Although several EP300-targeting HAT inhibitors including compounds optimized for oral administration are currently under development, none have yet progressed to clinical application due to limitations such as low specificity and potential off-target effects. High-lactate tumors are closely associated with hypoxia and an immunosuppressive tumor microenvironment. 6 Advances in antibody–drug conjugates 52 and strategies that modulate the tumor immune microenvironment 53 may offer opportunities to selectively deliver these inhibitors to high-lactate tumors. Therefore, the development of clinically applicable ENO2 or HAT inhibitors targeting EP300 via multiple delivery strategies is warranted. In summary, high H4K5la expression was identified as an independent prognostic factor for patients with HCC. We also demonstrate a novel mechanism by which H4K5la promotes HCC progression through the upregulation of ENO2 and the establishment of a positive feedback loop that accelerates glycolysis. Disruption of this metabolic feedback loop may suppress tumor growth and augment the efficacy of existing HCC therapies, thereby representing a promising therapeutic strategy. Abbreviations ChIP, chromatin immunoprecipitation; DFS, disease-free survival; H3K18la, histone H3 lysine 18 lactylation; H4K5ac, histone H4 lysine 5 acetylation; H4K5la, histone H4 lysine 5 lactylation; HAT, histone acetyltransferase; HCC, hepatocellular carcinoma; HDAC, histone deacetylase; IHC, immunohistochemistry; KD, knockdown; LDH, lactate dehydrogenase; OE, overexpression; OS, overall survival; panKla, global lactylated lysine; PEP, phosphoenolpyruvate; TS, total score. Authors’ contributions Conceptualization and methodology: YA, STanaka, SS Verification and formal analysis: MY, YA, KO, KK, YT, STsukihara, TT, AK, KU, KY, AN, SM, HT. Software and data curation: SS, SW, KO. Investigation: KA, SW, DB. Funding acquisition: STanaka, YA, SS, MH. Writing original draft: MY. Writing, review, and editing: MY, YA, DB, STanaka. Supervision: YA, STanaka. Data availability The raw and processed sequencing data generated in this study have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession numbers GSE314769 (ChIP-seq) and GSE314771 (RNA-seq). All other data supporting the findings of this study are included within the article and its supplementary materials. Financial support This work was supported by Grants-in-Aid for Scientific Research (A, JP19H01055; B, JP23H02979, JP23K27670, JP24K02320) and Challenging Research (Exploratory, JP22K19554, JP25K22639, JP25K22573) from the Japan Society for the Promotion of Science (JSPS) KAKENHI; a research grant from the Princess Takamatsu Cancer Research Fund; and P-CREATE (JP19cm0106540) and the Program for Basic and Clinical Research on Hepatitis (JP23fk0210090, JP24fk0210102, JP24fk0210106, JP25fk0210136, JP25fk0210149, JP25fk0210163, JP25fk0310546, 25fk0210167) from the Japan Agency for Medical Research and Development (AMED). Conflict of interest The authors declare no conflicts of interest pertaining to this manuscript. Please refer to the accompanying ICMJE disclosure forms for further details. Acknowledgements We wish to thank BioRender ( https://www.biorender.com ) for figure creation and Ms. Hiromi Onari for her clerical assistance. We would like to thank Editage ( www.editage.jp ) for English language editing. Footnotes Author names in bold designate shared co-first authorship Supplementary data to this article can be found online at https://doi.org/10.1016/j.jhepr.2026.101786 . Contributor Information Yoshimitsu Akiyama, Email: yakiyama.monc@tmd.ac.jp. Shinji Tanaka, Email: tanaka.monc@tmd.ac.jp. Supplementary data The following are the Supplementary data to this article: Multimedia component 1 mmc1.pdf (3MB, pdf) Multimedia component 2 mmc2.docx (55.3KB, docx) Multimedia component 3 mmc3.pdf (807.5KB, pdf) Multimedia component 4 mmc4.pdf (19.4MB, pdf) References 1. 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