Novel histone modifications and liver cancer: emerging frontiers in epigenetic regulation

Abstract

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, and its onset and progression are closely associated with epigenetic modifications, particularly post-translational modifications of histones (HPTMs). In recent years, advances in mass spectrometry (MS) have revealed a series of novel HPTMs, including succinylation (Ksuc), citrullination (Kcit), butyrylation (Kbhb), lactylation (Kla), crotonylation (Kcr), and 2-hydroxyisobutyrylation (Khib). These modifications not only expand the histone code but also play significant roles in key carcinogenic processes such as tumor proliferation, metastasis, and metabolic reprogramming in HCC. This review provides the first comprehensive analysis of the impact of novel HPTMs on gene expression, cellular metabolism, immune evasion, and the tumor microenvironment. It specifically focuses on their roles in promoting tumor stem cell characteristics, epithelial–mesenchymal transition (EMT), and therapeutic resistance. Additionally, the review highlights the dynamic regulation of these modifications by specific enzymes, including “writers,” “readers,” and “erasers.”

Introduction

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide, characterized by a high incidence and resistance to both radiotherapy and chemotherapy. Despite surgical resection or ablation, up to 70% of patients experience tumor recurrence within five years [1]. Global statistics show that liver cancer accounts for 8.2% of cancer-related deaths, with annual mortality rates ranging from 250,000 to 1 million [2,3,4,5]. In the onset and progression of HCC, HPTMs serve as a crucial epigenetic regulatory mechanism. By dynamically modulating chromatin structure and gene expression, HPTMs play a central role in tumor proliferation, invasion, and metabolic reprogramming.

HPTMs regulate chromatin conformation and gene expression by dynamically modifying chromatin structure and influencing gene activity. These modifications play a crucial role in controlling cellular processes [6]. Traditional modifications, such as acetylation and methylation, have been well-established as playing significant roles in the development of HCC. In recent years, with the advancement of mass spectrometry (MS) technology [7], a series of novel HPTMS have been discovered, including succinylation (Ksuc), citrullination (Kcit), butyrylation (Kbhb), lactylation (Kla), crotonylation (Kcr), and 2-hydroxyisobutyrylation (Khib), which significantly expand the histone code. As summarized in Fig. 1, these modifications are dynamically regulated by specific enzymes categorized as “writers,” “readers,” and “erasers.” For example, writers such as lysine acetyltransferase 2A (KAT2A) and p300 catalyze the addition of these modifications, while readers like bromodomain-containing proteins interpret the marks, and erasers such as Sirtuin 5 (SIRT5) and histone acetyltransferase 1 (HDAC1) remove them. This highlights their functional diversity and underscores their significance in chromatin dynamics and epigenetic regulation in HCC.

Fig. 1

Metabolism-related diagram of novel histone modifications in HCC. This schematic highlights the interplay between various novel HPTMs and their corresponding metabolic pathways, focusing on the liver and its role in cancer progression. The diagram displays multiple types of acylation modifications, including ksuc, kcit, kbnb, kla, kcr, and khib, each modulated by specific “writers,” “erasers,” and “readers.” These metabolic intermediates, such as lactyl-CoA, succinyl-CoA, and crotonyl-CoA, originate from core metabolic processes and are critical for chromatin regulation

Studies have shown that these novel HPTMs exert a profound impact on tumor cell proliferation, migration, and invasion in HCC by regulating mechanisms such as metabolic reprogramming, the tumor microenvironment, and immune evasion. For example, Kla is considered metabolic-epigenetic bridges that connect cellular metabolic states with chromatin dynamics, while Kbhb and Ksuc play regulatory roles in immune responses and viral replication, providing new perspectives for the prevention and treatment of HCC.

This review aims to systematically summarize the latest research progress on HPTMs in HCC, with a focus on the molecular mechanisms of novel modifications and their functional roles in tumor initiation and progression. Through an in-depth analysis of these novel HPTMs, we hope to provide new perspectives for future basic research and clinical translation, as well as contribute to the development of personalized diagnostic and therapeutic strategies for HCC.

Histone post-translational modifications

Histones are highly conserved basic proteins predominantly found in the nucleus of eukaryotic cells. Serving as the fundamental structural units of chromatin, they compact DNA tightly within the nucleus. Histones are categorized into two types: core histones (H2A, H2B, H3, and H4) and the linker histone ( H1), as illustrated in Fig. 2. Core histones form an octamer, around which DNA wraps approximately 1.65 times to generate nucleosomes, the fundamental structural units of chromatin. The linker histone binds to nucleosomes and linker DNA, enhancing nucleosome stability and facilitating chromatin compaction [8].

Fig. 2

Novel HPTMs and their roles in HCC. The schematic illustrates six novel histone modifications in HCC, highlighting the novel histone sites associated with HCC progression and their critical roles in regulatory pathways. Additionally, it describes potential therapeutic targets or targeted drugs for these novel HPTMs, offering new perspectives and potential strategies for the personalized treatment of HCC

The traditional HPTMs associated with HCC primarily include methylation, phosphorylation, ubiquitination, and acetylation [9]. HPTMs, such as acetylation and methylation, regulate chromatin compaction and affect the recruitment of remodeling complexes and transcription factors, thereby directly controlling gene transcription. These modifications play a critical role in tumor initiation and progression, while also participating in the regulation of key cellular processes, including DNA repair, DNA replication, and gene expression. In cancer, abnormal methylation of CpG islands is a key mechanism for the silencing of chromatin in promoter regions [10,11,12]. DNA methyltransferases (DNMTs) add methyl groups to the CpG islands in the promoter regions, which attract methyl-binding proteins (MBPs, such as MeCP2). These MBPs act as "bridges" to further recruit histone deacetylases (HDACs) and histone methyltransferases (HMTs). These enzymes remove histone acetyl groups and add repressive methylation marks (e.g., H3K9me3), leading to a highly compacted chromatin structure that prevents the binding of transcription factors [13]. For example, hypermethylation of the p16 promoter region silences its expression, disrupting its regulatory role in the cell cycle and promoting abnormal cell proliferation. This epigenetic alteration often occurs in the early stages of cancer and can also be observed in precancerous lesions [14]. Similarly, the silencing of the DNA repair gene MGMT(O6-methylguanine-DNA methyltransferase) reduces DNA repair capacity, increases genomic instability, and provides opportunities for tumorigenesis [15,16,17].

Therapeutic strategies targeting these epigenetic mechanisms have shown great potential in HCC. HDAC inhibitors (e.g., Panobinostat and Belinostat) restore the expression of silenced tumor suppressor genes by inhibiting HDAC activity, significantly suppressing cell proliferation and angiogenesis in HCC. Additionally, the EZH2 (enhancer of zeste homolog 2) inhibitor GSK126, as a histone methyltransferase (HMT) inhibitor, reduces H3K27me3 marks by inhibiting EZH2 activity, reactivates the expression of tumor suppressor genes, promotes tumor cell apoptosis, and enhances the cytotoxic activity of immune cells against the tumor [3, 18,19,20].

While HPTMs have been extensively studied, advancing our understanding of gene expression regulation and HCC-related mechanisms, emerging evidence indicates that the diversity of HPTMS extends well beyond these established types. Ongoing advances in epigenetic research have identified numerous novel HPTMs, including succinylation (ksuc), citrullination (kcit), hydroxybutyrylation (kbnb), lactylation (kla), crotonylation (kcr), and 2-hydroxyisobutyrylation (khib). These modifications not only provide new insights into gene regulation but also play distinct roles in the onset and progression of HCC. A comprehensive summary of these mechanisms and their impacts is provided in Fig. 2. Novel HPTMs act as crucial players in HCC, paving the way for new research and therapeutic strategies.

Succinylation

Ksuc is a novel HPTM that has garnered significant attention in recent years. This modification entails the covalent attachment of a succinyl group to the lysine residues of histones, thereby altering their physicochemical properties and subsequently influencing chromatin structure and gene expression [21, 22]. This modification was first identified by Zhang et al. in 2011 using MS analysis [21]. Ksuc occurs in both bacterial and mammalian cells. The primary enzymes catalyzing Ksuc include P300/CREB-binding protein (P300/CBP), KAT2A, and histone acetyltransferase 1 (HAT1). Additionally, SIRT5 and Sirtuin 7 (SIRT7) have been identified as the main desuccinylase enzymes. The YEATS domain of the GAS41 protein specifically recognizes Ksuc and functions as a reader protein, participating in associated regulatory processes [23,24,25,26].

Guang Yang et al. [26] reported that HAT1 expression is significantly upregulated in HCC. HAT1 not only functions as an acetyltransferase but also possesses sucryltransferase activity, transferring the succinyl group to the lysine 122 (K122) site on histone H3, forming H3K122suc. H3K122suc is a biologically significant novel HPTM, primarily catalyzed by HAT1. H3K122suc, located at the nucleosomal DNA contact interface, reduces histone–DNA binding affinity, leading to chromatin decondensation and enhanced transcriptional activity at gene promoter regions. In cancer cells, this modification provides epigenetic support for tumor cells by enhancing the expression of tumor-associated genes, such as CREBBP and BPTF. It promotes cell proliferation, metabolic reprogramming, and invasion. Furthermore, H3K122suc exerts a profound impact on tumor growth by directly regulating the transcriptional activation of glycolysis-related genes (e.g., PGAM1) and significantly enhancing PGAM1 activity through sucrylation at its K99 site. This drives metabolic reprogramming in cancer cells, increasing glycolytic flux, ATP production, and the supply of metabolic intermediates to support rapid cell proliferation and metabolic demands. Experimental studies have shown that knockout of HAT1 or inhibition of H3K122sucrylation effectively blocks cancer cell proliferation and metabolic activity.

HAT1 is highly expressed in various cancers, including liver, pancreatic, and cholangiocarcinoma, and is closely associated with tumor progression. These findings highlight the critical role of HAT1-mediated H3K122suc in epigenetic regulation and tumor metabolism. Therefore, H3K122suc and its catalytic enzyme HAT1 not only reveal the core mechanisms underlying gene regulation and tumorigenesis but also provide promising potential targets for anticancer therapy [26, 27].

KAT2A (also known as GCN5) was initially identified as a histone acetyltransferase, capable of catalyzing the acetylation of H3K9 in the TGFB1 promoter region, thereby activating TGFB1 expression and promoting the secretion of large amounts of TGF-β by tumor cells. TGF-β induces the differentiation of CD4 + T cells into Treg cells, which in turn secrete immune-suppressive factors such as IL-10 and TGF-β. These factors inhibit the activity of CD8 + T cells, weakening their ability to kill tumor cells. At the same time, TGF-β induces CD8 + T cells to overexpress immune checkpoint molecules such as PD-1 and CTLA-4, leading to T cell exhaustion. Ultimately, this promotes immune evasion in liver tumor cells, creating favorable conditions for tumor cell proliferation and metastasis [28].

Recent studies, however, have shown that KAT2A not only possesses acetyltransferase activity but also exhibits succinyltransferase activity. It can transfer the succinyl group from succinyl-CoA to lysine 79 (H3K79) of histone H3, resulting in the formation of H3K79suc. KAT2A boosts its succinyltransferase activity by forming a supramolecular complex with α-ketoglutarate dehydrogenase (α-KGDH), thereby locally generating high concentrations of succinyl-CoA in the nucleus. This local generation of succinyl-CoA significantly increases the efficiency of KAT2A in catalyzing H3K79suc, enabling it to effectively catalyze the modification even under low concentrations of succinyl-CoA. The H3K79suc modification reduces the binding affinity between histones and DNA, leading to chromatin decompaction and activation of tumor-related gene transcription. Notably, in tumor cells, this modification also enhances the expression of genes involved in cell cycle regulation and metabolic reprogramming, thereby supporting the rapid proliferation of tumor cells [24]. However, it is unfortunate that no study has yet clearly defined how KAT2A-mediated Ksuc specifically influences tumorigenesis and progression in HCC.

KAT2A-mediated Ksuc plays a crucial role in the development of chronic hepatitis B (HBV), which is one of the major etiological factors for HCC. HBV is a hepatotropic DNA virus, and its encoded gene products are essential for viral replication. Among these, covalently closed circular DNA (cccDNA) serves as both the template for HBV transcription and the core of viral replication [29, 30]. Studies have shown that KAT2A interacts with the HBV core protein and catalyzes the H3K79suc on cccDNA, thereby enhancing the transcriptional activity of cccDNA and promoting HBV replication. Furthermore, research by Yuan et al. found that interferon α (IFN-α) significantly inhibits the activity of HBV cccDNA by downregulating KAT2A-mediated H3K79succ [31, 32]. These findings reveal the critical regulatory role of ksuc in the HBV life cycle and provide new insights for targeted therapies against HBV and the prevention of HCC.

Citrullination

Kcit, also referred to as deimination, is catalyzed by peptidylarginine deiminase (PAD), which convert arginine residues into citrulline. This modification alters the charge state of proteins and is primarily associated with the regulation of gene expression and immune responses [33, 34]. Approximately 10% of all histone molecules are Kcit, highlighting the critical role of this post-translational modification in various nuclear processes [35]. Another well-known function of kcit is its role in triggering the formation of neutrophil extracellular traps (NETs), a mechanism used to immobilize and eliminate bacterial pathogens [36, 37]. The primary writers of kcit are members of the PAD enzyme family, particularly PAD4. This enzyme converts arginine residues in histones H3 and H4 to citrulline, thereby regulating chromatin structure and gene expression. Currently, no identified "erasers" can reverse Kcit, suggesting that once this modification occurs, it is generally regarded as irreversible [38]. Furthermore, no "reader" proteins specifically recognizing and binding to Kcit have been clearly identified. There is a dearth of literature on the influence of kcit on HCC biology and progression. In this article, I summarize and critically review the published studies on this topic.

Through ELISA analysis, studies [39] revealed significantly higher levels of H3Cit in HCC tissues (72.25 ng/mg) compared to non-tumor tissues (44.02 ng/mg). In HCC samples with high H3Cit expression, Beclin1 mRNA levels were also notably elevated. Specifically, the average Beclin1 mRNA level was 5.772 in the high H3Cit group, compared to 0.552 in the low H3Cit group. Regression analysis further confirmed a significant positive correlation between H3Cit and Beclin1 mRNA expression (R² = 0.797, p < 0.0001). Moreover, elevated H3Cit and Beclin1 expression were strongly associated with vascular invasion, higher alpha-fetoprotein (AFP) levels, and advanced TNM staging in HCC. Patients with high H3Cit and Beclin1 mRNA expression were more likely to exhibit vascular invasion, and serum AFP levels correlated positively with both H3Cit and Beclin1 mRNA expression. In patients with advanced TNM stages (e.g., stage III), H3Cit and Beclin1 expression were significantly elevated. Notably, patients with high expression of both H3Cit and Beclin1 had significantly shorter median survival, with 19 months for the high-expression group versus 30 months for the low-expression group.

Further studies indicate that H3Cit regulates Beclin1 mRNA expression by altering chromatin structure or recruiting co-activator complexes, thereby influencing Beclin1 protein levels. As a central regulator of the autophagy pathway, Beclin1 promotes the expression of peptidylarginine deiminase type 4 (PADI4), a calcium-dependent enzyme. Elevated PADI4 expression in HCC patients undergoing transarterial chemoembolization (TACE) is associated with chemotherapy resistance, as it induces autophagy to help tumor cells cope with the adverse microenvironment and chemotherapy-induced stress. Experiments showed that specific knockdown of Beclin1 significantly diminished PADI4-induced autophagy, enhancing the sensitivity of HCC cells to chemotherapy. Therefore, targeting H3Cit or Beclin1 may represent a promising therapeutic strategy to improve the efficacy of HCC treatment [39, 40].

H3Cit is a critical initiating step in the formation of neutrophil extracellular traps (NETs). During NET formation, H3Cit removes the positive charge from H3 arginine residues, significantly decreasing chromatin compaction, leading to chromatin decondensation and subsequent release into the extracellular space, where it forms NETs in association with myeloperoxidase (MPO) and neutrophil elastase [41]. In HCC, H3Cit levels are significantly elevated and directly contribute to the remodeling of the tumor microenvironment by promoting NET formation. H3Cit-mediated NETs enhance tumor progression by inducing angiogenesis, increasing the invasion and migration abilities of cancer cells, and suppressing the activity of effector T cells. Additionally, H3Cit exacerbates inflammation, creating a pro-inflammatory and pro-tumor feedback loop that further supports HCC development [42, 43].

In summary, H3Cit, a novel HPTM, has been receiving increasing attention for its role in HCC. H3Cit levels are significantly elevated in HCC tissues and strongly correlate with the expression of Beclin1 mRNA. By regulating Beclin1, H3Cit promotes autophagy activity, helping tumor cells cope with chemotherapy-induced stress and enhancing chemotherapy resistance. Furthermore, H3Cit induces the formation of NETs, which further promote inflammation and the proliferation and metastasis of tumor cells. Overall, H3Cit plays a critical role in the onset and progression of HCC and may influence the tumor microenvironment through multiple mechanisms, such as autophagy and NET formation. Therefore, targeting H3Cit or its associated regulatory factors may offer a novel strategy for future HCC treatment.

β-Hydroxybutyrylation

Kbhb is a novel HPTM, first discovered by Xie et al. in [44]. This modification entails the chemical attachment of the β-hydroxybutyryl group, derived from β-hydroxybutyrate (BHB), to lysine residues on histones. It typically occurs during significant changes in cellular metabolic states, such as fasting or conditions like diabetes. Currently, P300/CBP is recognized as the primary writer of kbhb [44]. The primary enzymes responsible for reversing kbhb are SIRT3, SIRT5, and HDAC3. Currently, no reader proteins specifically recognizing Kbhb have been definitively identified. However, evidence suggests that reader proteins associated with other HPTMs may also recognize kbhb. For example, proteins from the bromodomain family might interact with this modification, thereby regulating downstream gene expression [45].

MTA2 (metastasis-associated protein 2) is a critical component of the NuRD (nucleosome remodeling and histone deacetylase) complex and belongs to the MTA family. MTA2 has been shown to regulate HCC cell proliferation and invasion by influencing Kbhb. Kbhb primarily accumulates at the transcriptional start sites (TSS) of genes, and its activation of gene expression is closely associated with cancer progression. In HCC, Kbhb levels are significantly elevated, particularly the enrichment of H3K9bhb at the promoters of oncogenes such as JMJD6, GREB3, and NPM1, which enhances their expression [44, 46].

Specifically, MTA2 inhibits the expression of BDH1 by forming R-loop structures. BDH1 is a key enzyme in ketone body metabolism, catalyzing the interconversion between acetoacetate (AcAc) and β-hydroxybutyrate (βHB). When BDH1 expression is suppressed, intracellular βHB accumulates, driving the enrichment of H3K9bhb. Experimental data show that in MTA2-overexpressing HCC cells, H3K9bhb levels increase 2.3-fold compared to controls, and the expression of stemness markers, including CD44, SOX9, and EpCAM, is markedly elevated. Additionally, clonogenic and spheroid-forming abilities are increased by approximately 2.5-fold and threefold, respectively. Restoration of BDH1 expression or treatment with RNase H1 to eliminate R-loops resulted in a more than 50% reduction in H3K9bhb levels, significantly suppressing the oncogenic phenotype. Further animal experiments revealed that in a diethylnitrosamine (DEN)-induced HCC mouse model, MTA2 overexpression led to a significant increase in hepatic tumor burden compared to controls, while BDH1 restoration reversed this effect. Additionally, clinical analysis of 340 HCC patients revealed a significant correlation between high expression of MTA2 and H3K9bhb with poor prognosis. The median overall survival was only 18 months for the MTA2 high-expression group, compared to 33 months for the low-expression group (p < 0.05) [46]. These findings highlight the critical role of the Kbhb-mediated MTA2-BDH1 axis in HCC and suggest that targeting this pathway could provide a new therapeutic strategy for HCC.

The ketogenic diet (KD) is a high-fat, low-carbohydrate, and moderate-protein dietary regimen that has been used in clinical practice since the 1920s [47]. Its anticancer potential is primarily attributed to the interference with tumor metabolism (e.g., lowering blood glucose and insulin levels), thereby inhibiting the proliferation and differentiation of tumor cells.

A meta-analysis demonstrated that the KD can delay tumor growth and improve survival in mice by altering the metabolic environment. The survival time ratio (MR) for the KD group was 0.85 (95% HPDI: 0.73–0.97), indicating a 15% increase in survival compared to the standard diet (SD) group. When the ketogenic diet was initiated immediately after tumor implantation, the MR was 0.80 (95% HPDI: 0.69–0.92), showing significant anticancer effects [48].

KD promotes the conversion of free fatty acids in the liver into ketone bodies (including acetoacetate, βHB, and acetone) by reducing glucose utilization, with βHB being the predominant ketone body. For instance, in an experiment, after KD treatment, the serum βHB concentration in mice increased from 0.3 mM in the control group to 10 mM (P < 1 × 10⁻¹⁵) [49]. KD significantly increases βHB levels in vivo and promotes Kbhb, altering histone functions and consequently affecting the expression of metabolism-related genes in HCC. MS analysis revealed that the levels of Kbhb at the H3K9, H3K18, and H4K5 sites were significantly elevated in the KD treatment group compared to the control group (Kbhb increased approximately sixfold, P < 0.01). These modifications were notably enriched in the promoter regions of metabolism-related genes, leading to transcriptional repression. For instance, in the KD group, the mRNA levels of aldolase B (ALDOB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and isocitrate dehydrogenase 2(IDH2) were significantly lower than those in the control group (P < 0.01). The downregulation of these metabolic genes directly impacted glycolysis and the tricarboxylic acid (TCA) cycle,, leading to a reduced energy supply for HCC cells and impairing their proliferation and migration. Additionally, KD mediates negative regulation of signaling pathways through H3K9bhb modifications. Specifically, in the mTOR signaling pathway, the increase in H3K9bhb downregulates both upstream and downstream signals, including the phosphorylation of key effector proteins such as S6K and 4EBP1. This mechanism deprives liver cancer cells of essential energy and growth signals, thereby significantly inhibiting their proliferative capacity [50, 51].

The accumulation of Kbhb plays a crucial role in the initiation and progression of HCC. MTA2 inhibits BDH1 expression, leading to the accumulation of βHB, which in turn promotes the modification of H3K9bhb, enhancing tumor cell proliferation and stemness. Furthermore, the KD increases βHB levels, thereby boosting Kbhb and suppressing the expression of metabolism-related genes, which disrupts HCC cell metabolism and inhibits their proliferation. These findings highlight the pivotal role of Kbhb in HCC, suggesting that targeting this pathway could offer new therapeutic strategies for liver cancer treatment.

Lactylation

Kla is a recently identified HPTM. This modification is initiated by the accumulation of lactate in tumor cells, with intracellular lactate concentration showing a positive correlation with kla levels [52]. Kla was first proposed by Zhang et al. in a 2019 study [53]. Lactate, as a metabolic byproduct, not only plays a role in cellular metabolism but also directly participates in epigenetic regulation. P300/CBP is currently recognized as the primary writer of kla, catalyzing the formation of this modification by transferring lactyl groups derived from the lactate accumulated in cells. To date, the erasers and readers of kla have not yet been identified [53,54,55].

Kla plays a crucial role in the onset and progression of HCC by regulating metabolism and gene expression. Kla modifies chromatin structure, opening gene promoter regions and significantly enhancing the binding efficiency of transcription factors to DNA, thereby promoting gene expression. ATAC-seq analysis revealed that chromatin accessibility in gene promoter regions enriched with H3K9la was significantly higher than in unmodified regions. This chromatin opening mechanism enhances the expression of genes related to lactate metabolism (e.g., LDHA, PDK1, SLC16A3), cell cycle regulation (e.g., Cyclin D1), and oncogenes (e.g., Endothelial Cell-Specific Molecule 1, ESM1). High expression of lactate metabolism genes leads to increased lactate accumulation, which not only serves as a metabolic substrate to drive the tricarboxylic acid (TCA) cycle but also promotes Kla, activating the expression of genes associated with tumor proliferation, migration, and resistance. Cyclin D1 is a key regulator of the cell cycle, working in conjunction with CDK2 and Cyclin E1 to regulate the G1/S-phase transition. In HCC cells with high Cyclin D1 expression, the cell cycle is accelerated, proliferation is significantly enhanced, and the malignant phenotype of the tumor cells is promoted. High expression of ESM1 accelerates the epithelial-to-mesenchymal transition (EMT) process. EMT refers to the process in which cells transition from an epithelial phenotype to a mesenchymal phenotype under specific physiological or pathological conditions. During this process, epithelial cells lose their characteristic polarity and intercellular tight junctions, while acquiring features typical of mesenchymal cells, such as enhanced migratory and invasive abilities.

In samples with high H3K9la, Cyclin D1 mRNA expression was significantly elevated compared to low-modification samples. Treatment of HCC cells with the p300 inhibitor A485 resulted in a 56% reduction in H3K9la levels and a 43% reduction in H3K56la levels. Additionally, the expression of lactate metabolism genes, such as LDHA, was significantly decreased (P < 0.01). Inhibition of Kla using 2-deoxy-D-glucose (2-DG) resulted in Western blot and immunofluorescence analyses showing a significant increase in E-cadherin expression and a decrease in N-cadherin expression, effectively inhibiting the EMT process (P < 0.01). In the mouse xenograft model, 2-DG treatment significantly reduced tumor growth and metastasis, with a notable decrease in tumor size and volume (P < 0.01) [56, 57].

Additionally, multiplexed immunohistochemistry (mIHC) analysis revealed that in HCC tumor regions with high GPC3 + panKla + density, the levels of H3K18la and H3K27la were approximately 2.3-fold and 1.8-fold higher, respectively, compared to low-density samples. Spearman correlation analysis showed a significant positive correlation between the expression of GPC3 + panKla + and H3K18la levels (P < 0.05). GPC3 + panKla + serves as a malignant feature marker for highly proliferative tumor cells in HCC. This finding suggests that Kla is not only a key regulator of HCC metabolism and gene expression but may also serve as a potential biomarker for assessing HCC progression and prognosis [53, 58].

In summary, Kla creates a metabolic-epigenetic feedback loop in HCC through mechanisms such as metabolic remodeling and chromatin opening. This loop significantly enhances the expression of lactate metabolism genes and oncogenes, promoting tumor proliferation and migration. These modification levels not only serve as key driving factors in HCC initiation and progression but also hold potential as molecular biomarkers for diagnosis and prognosis. Additionally, targeting the regulatory factors of Kla modifications (e.g., p300) or key enzymes in lactate metabolism (e.g., LDHA) may offer novel therapeutic strategies for HCC treatment.

Tumor cells frequently depend on enhanced glycolysis, a metabolic shift known as the Warburg effect, which remains active even under sufficient oxygen conditions. This metabolic preference results in elevated lactate production [59, 60]. Given the central role of the Warburg effect in cancer metabolism, it also presents an attractive target for therapeutic intervention [61]. One of the key breakthroughs in this area comes from Lianhong Pan et al., who demonstrated that Demethylzeylasteral (DML), a triterpenoid compound with antitumor properties, significantly inhibits the proliferation and migration of liver cancer stem cells (LCSCs) by reducing lactate levels and subsequently suppressing H3la. This finding was further validated in a nude mouse xenograft model, highlighting the potential of DML as a novel therapeutic agent against HCC. By targeting lactate metabolism and kla, DML offers a promising new approach to HCC treatment [56]. Additionally, to validate the impact of kla on HCC, Peng Zhao et al. employed 2-deoxy-D-glucose(2-DG), a glycolysis inhibitor, in a mouse model to reduce lactate production and thereby suppress kla [57]. The results demonstrated that 2-DG not only significantly reduced tumor volume but also effectively inhibited tumor metastasis.

Similarly, studies have shown that royal jelly acid (RJA), a natural compound, disrupts the glycolytic pathway in HCC, significantly reducing intracellular lactate levels and inhibiting H3K9la and H3K14la. This effectively suppresses the proliferation, migration, and invasion of HCC cells while promoting apoptosis. In animal studies with a xenograft mouse model, treatment with 20 mg/kg RJA resulted in a nearly 50% reduction in tumor weight compared to the control group. Moreover, Ki67 expression in tumor tissues was significantly reduced, indicating that RJA effectively inhibits tumor cell proliferation. In addition, RJA demonstrated significant antitumor activity against HCC cells, such as Hep3B and HCCLM3, with IC50 values of 27.35 μM and 23.66 μM, respectively. Treatment with 20 μM RJA resulted in a 28.01% and 20.81% reduction in the S-phase proportion of Hep3B and HCCLM3 cells, respectively. Furthermore, RJA upregulated the expression of pro-apoptotic proteins BAX(Bcl-2-associated X protein) and Caspase 8 while downregulating the anti-apoptotic protein BCL2 (B cell lymphoma 2), significantly increasing the BAX/BCL2 ratio and further inducing apoptosis [62]. RJA exhibits significant antitumor activity by modulating glycolytic metabolism, inhibiting kla, and inducing apoptosis. These findings not only highlight the critical role of Kla in HCC progression but also suggest that RJA has potential as a novel therapeutic candidate for HCC, offering a new strategy for the treatment of HCC.

In conclusion, Kla plays a key role in HCC by regulating metabolism and gene expression. It enhances the expression of genes associated with lactate metabolism, cell cycle, and tumor progression by opening chromatin structure, significantly promoting tumor proliferation and migration. Inhibition of Kla formation not only effectively suppresses tumor cell proliferation and migration but also holds potential as a biomarker for prognostic assessment of HCC progression. Furthermore, targeting Kla-related regulatory factors (e.g., P300) or lactate dehydrogenases (e.g., LDHA) offers new potential strategies for HCC treatment. Natural compounds such as DML and RJA, by modulating lactate metabolism and Kla modifications, exhibit significant antitumor effects, paving the way for new clinical applications in HCC therapy.

Crotonylation

Kcr is a novel HPTM, first identified by Tan et al. [63]. In this study, the researchers identified 28 Kcr sites, including H3 and H4, using MS. Among these, H3K18 is highlighted as a key regulatory modification. These sites are distributed across various histones and are closely associated with active gene promoters and potential enhancers. Kcr primarily occurs on the ε-amino group of lysine, and its unique planar structure, along with the four-carbon chain length, differentiates it from lysine acetylation. Similar to acetylation, kcr can also modulates histone–DNA interactions, impacting chromatin architecture and gene regulation. Variations in Kcr levels are associated with tumor cell aggressiveness and proliferative capacity across different cancers, indicating its critical role in oncogenesis and metastasis. Kcr is catalyzed by specific “writers,” including p300/CBP, KAT2A, and the MYST family, which introduce crotonyl groups to lysine residues on histones [45]. Its biological function is primarily mediated by “reader” proteins, including bromodomain-containing protein 4 (BRD4), YEATS, double PHD fingers domain (DPF), and chromodomain Y-like protein (CDYL). These recognition modules bind to crotonyl groups, further influencing gene expression and chromatin structure, thereby exerting their functions [64, 65]. Meanwhile, deacetylases, including HDAC1-3 and SIRT1-3, function as “erasers” capable of removing Kcr, thereby regulating this dynamic process [66,67,68].

Immunohistochemical staining was employed to assess kcr expression levels across various human tumor tissues, revealing a significant reduction in kcr levels in HCC tissues. This decrease suggests that kcr may be linked to tumor suppression in HCC. Further analysis of 68 HCC samples demonstrated a close correlation between kcr expression levels and TNM staging. Specifically, lower kcr expression was associated with higher TNM stages, indicating more advanced liver cancer. Moreover, the study found that knockdown of HDAC1 and HDAC3 [69,70,71] or treatment with the HDAC inhibitor Trichostatin A, significantly increased kcr levels in liver cancer cells, which in turn inhibited the migratory capacity of these cells. These results indicate that the inhibition of HDAC1 and HDAC3 reduces the invasiveness of liver cancer cells, highlighting the critical role of kcr regulation in liver cancer invasion and metastasis. The effects of kcr on HCC proliferation were also investigated. Studies have shown that H3K27cr possesses a unique “gene pause” function, which plays a crucial role in regulating tumor suppressor genes by activating their expression to inhibit cancer cell proliferation. Conversely, decreased H3K27cr levels lead to the silencing of tumor suppressor genes, accelerating cancer progression. Further research demonstrated that increasing Kcr levels through HDAC inhibition significantly suppresses HCC proliferation. These findings suggest that modulating H3K27cr dynamics—by activating specific crotonyltransferases or inhibiting the de-crotonylase enzyme SIRT6—could become a promising strategy for controlling cancer cell growth, offering new potential for cancer therapy [72, 73].

Kcr, recognized as a tumor suppressor in liver cancer development, constitutes a significant type of histone modification involved in HCC progression and presents a wide avenue for future research. However, our understanding of the potential mechanisms underlying kcr’s role in HCC progression remains incomplete. Future investigations should prioritize elucidating the specific mechanisms through which kcr influences the advancement of liver cancer.

2-Hydroxyisobutyrylation

Discovered in 2014 through MS, Khib is a novel histone modification that pervades both the N-terminal tails and core regions of histones, directly influencing chromatin structure [74]. Khib neutralizes the positive charge of lysine residues, altering their electrostatic properties and preventing strong electrostatic interactions with negatively charged molecules, such as the DNA phosphate backbone. This modification disrupts the electrostatic interaction between lysine and DNA. Meanwhile, the hydroxyl group enhances the intermolecular hydrogen bonding network, providing new hydrogen bond donor or acceptor sites, which facilitates chromatin relaxation and subsequently activates gene transcription. By linking dynamic changes in Khib to cellular metabolic states, it establishes Khib as a metabolic-epigenetic bridge in gene regulation. Khib’s conservation across diverse eukaryotes, with pronounced roles in transcriptionally active regions, underscores its broader significance in gene control. Its enrichment in chromatin regions associated with gene activity further suggests potential regulatory roles in diseases, including cancer and metabolic disorders [74]. The primary writers of khib include p300, Esa1p (in yeast), and Tip60 (in humans) [74, 75], which can catalyze khib both in vitro and in vivo. While specific readers of khib are still being investigated, bromodomain and extra-terminal domain (BET) proteins are known to potentially play a role in this process [76]. The primary erasers include HDAC1-3 and SIRT2 [66, 77], which remove khib modifications from histone H4, thereby regulating chromatin structure and gene expression.

Currently, research on the relationship between Khib and HCC remains limited, with existing studies primarily focused on the etiology of HCC. HBV infection is one of the major causative factors of HCC, and chronic HBV infection is closely associated with the onset and progression of HCC. In this context, the transcription and replication of cccDNA are crucial steps in maintaining chronic HBV infection, directly influencing the onset and progression of HCC. A study indicates that, in HBV-infected hepatocytes, IFN-α significantly enhances HDAC3 function by inducing the expression of interferon-stimulated genes (ISGs), leading to a reduction in H4K8 Khib levels on the cccDNA minichromosome. HDAC3 targets and binds to the cccDNA minichromosome, removing the Khib on H4K8, which induces the transition of cccDNA from an open, active state to a silent state. This significantly reduces the transcriptional activity of cccDNA and inhibits HBV replication. This mechanism has been validated in HCC cells (e.g., HepG2-NTCP and HepG2.2.15) as well as in human liver chimeric mouse models. Experimental results showed that overexpression of HDAC3 or treatment with IFN-α significantly reduced H4K8 Khib levels, decreased HBV transcriptional activity (assessed by the pgRNA/cccDNA ratio), and lowered key indicators of viral replication (HBV DNA, HBsAg, and HBeAg levels). This study provides the first evidence that H4K8 Khib is a critical epigenetic mark for the active transcription of HBV cccDNA. This modification promotes HBV transcription and replication by maintaining the open state of the minichromosome. HDAC3, by removing this modification, drives cccDNA transcription into a silent state, fundamentally altering the viral survival environment within the host cell. Furthermore, it was revealed that IFN-α inhibits HBV cccDNA transcription at the epigenetic level by promoting HDAC3-mediated H4K8 de-Khib. This suggests that IFN-α not only exerts its antiviral effects through traditional immune pathways but also directly intervenes in the epigenetic regulation of the virus, thereby broadening the scope of IFN-α as an antiviral therapeutic [78]. The study provides novel insights and a practical foundation for the precise diagnosis and biomarker development of chronic HBV infection. Furthermore, it offers new scientific evidence and directions for preventing HBV-induced HCC, delaying the progression of chronic hepatitis B, and optimizing combined antiviral and antitumor therapies.

Aflatoxin is a carcinogenic metabolite produced by Aspergillus species. Globally, it is responsible for approximately 28% of HCC cases, making it one of the primary etiological factors associated with this disease [79]. Jing Wang et al. [80] demonstrated that deletion or inactivation of the Afngg1 gene suppresses H4K5hib and H4K8hib, resulting in decreased expression of aflatoxin synthesis-related genes, such as aflC, aflX, and aflQ. This suppression consequently inhibits aflatoxin production. By reducing aflatoxin synthesis, the incidence of liver cancer can be effectively lowered, rendering this finding highly significant for clinical and research applications. Therefore, the role of khib in aflatoxin production presents a promising avenue for the development of prevention and treatment strategies for aflatoxin-induced liver cancer.

In summary, Khib, as a novel HPTM, plays a crucial role in gene expression regulation. It promotes chromatin relaxation and transcription activation by altering the charge characteristics of lysine residues and facilitating hydrogen bond network formation. The study also highlights the key role of Khib in HBV infection, where IFN-α regulates HDAC3 to remove Khib from H4K8, thereby inhibiting HBV transcription and replication. Additionally, Khib exerts significant regulatory effects in aflatoxin biosynthesis, suggesting its potential application in HCC. Although research on Khib in HCC is still in its early stages, its prominent position in epigenetic regulation provides a new direction for future disease prevention, diagnosis, and therapeutic strategies.

Conclusion

This review provides a comprehensive summary of the important roles of novel HPTMs in HCC, with an in-depth exploration of how these modifications regulate chromatin conformation, gene expression, and the tumor microenvironment. In recent years, with advances in MS technology, the discovery of novel HPTMs (e.g., kla, ksuc, and Kbhb) has revealed their multifaceted impact in HCC. These modifications not only drive the initiation and progression of HCC by regulating the expression and epigenetic state of tumor-associated genes, but also profoundly influence the metabolic reprogramming, tumor invasion, and immune regulation of HCC through their dynamic interplay with metabolic products.

Metabolic adaptation is one of the key features of HCC, forming a complex and dynamic feedback loop with epigenetics. In HCC, metabolic intermediates can directly drive the generation of novel HPTMs, thereby regulating chromatin structure and gene transcription to meet the metabolic demands of rapid proliferation. For example, lactate accumulation drives Kla, which regulates the expression of specific transcription factors and metabolic genes (e.g., LDHA and PDK1), thereby promoting immune evasion and angiogenesis within the tumor microenvironment, significantly accelerating tumor cell growth and metastasis. In addition, Kla enhances the expression of key glycolytic genes, further activating the glycolytic pathway, reinforcing the Warburg effect, and leading to increased lactate accumulation, thereby creating a positive feedback loop between metabolism and epigenetics. Additionally, BHB, as a key metabolic signaling molecule, regulates Kbhb to activate the expression of genes associated with tumor stemness and EMT, thereby conferring HCC cells with enhanced invasiveness and metastatic potential. These metabolism-driven HPTMs not only reflect the direct effects of metabolic products as substrates but also highlight the bidirectional regulatory relationship between metabolism.

Moreover, targeted therapies aimed at novel HPTMs have shown significant potential in HCC research. These modifications not only unveil the complex link between tumor metabolism and epigenetics but also provide new targets and strategies for treatment. For example, Kla drives the metabolic reprogramming of HCC by regulating glycolytic genes, while compounds such as DML significantly reduce tumor cell growth and migration by inhibiting kla. The dynamic regulatory mechanisms of novel HPTMs provide additional targets for precision therapy. For instance, the SIRT family of deacetylases, acting as "erasers," are involved in the regulation of Kcr and Kbhb, profoundly impacting tumor cell invasiveness and metabolic adaptability. By targeting these "erasers" or other regulatory factors, it becomes possible to further intervene in the expression of tumor-associated genes, thereby limiting HCC progression.

Although significant progress has been made in understanding the role of novel HPTMs in HCC, their molecular mechanisms, spatiotemporal specificity, and interactions with classical epigenetic marks require further exploration. Future research should focus on: developing high-resolution mass spectrometry to analyze HPTM profiles and dynamic regulation; assessing the therapeutic potential of HPTMs combined with metabolic regulation; and conducting large-scale clinical studies to evaluate HPTMs' value in HCC diagnosis and prognosis. A deeper understanding of these mechanisms could provide essential insights for precision diagnosis and personalized treatment of HCC.