Lactylation Modification and Esophageal Cancer: Research Progress From Hypoxia‐Induced Metabolic Reprogramming to Immune Escape

ABSTRACT

Esophageal cancer is a common malignancy of the digestive tract, marked by aggressive invasion, unfavorable prognosis, and limited responsiveness to existing therapeutic strategies. Hypoxia‐driven metabolic remodeling represents a key feature of the esophageal cancer microenvironment. In this context, enhanced glycolytic activity leads to lactate accumulation, thereby providing the metabolic substrate for lactylation. Lactylation, a recently recognized lysine post‐translational modification, has been detected on both histone and non‐histone proteins and regulates tumor biology by modulating chromatin accessibility, gene transcription, protein stability, and signaling pathway activity. Emerging evidence suggests that lactylation is closely involved in the malignant progression of esophageal cancer, including tumor cell proliferation, invasion, stemness maintenance, regulation of programmed cell death, immune escape, and treatment resistance. Under hypoxic conditions, increased lactate production may drive histone lactylation, including histone lactylation marks such as H3K9la, H3K18la, and H4K12la, as well as lactylation of non‐histone proteins. These modifications contribute to remodeling of the immunosuppressive tumor microenvironment and weaken anti‐tumor immune responses. This review highlights the biological basis of lactate metabolism and lactylation, focusing on their roles in metabolic reprogramming driven by hypoxia, the advancement of tumors, the regulation of the immune microenvironment, immune evasion, and resistance to therapy in esophageal cancer. Additionally, we explore the potential clinical significance of molecules associated with lactylation as diagnostic markers and therapeutic targets, offering fresh insights for precision treatment strategies in esophageal cancer.

Hypoxia‐induced metabolic reprogramming promotes glycolytic activity and lactate accumulation in esophageal cancer, thereby facilitating both histone and non‐histone lactylation. Through metabolic–epigenetic crosstalk, lactylation reshapes transcriptional programs associated with tumor proliferation, stemness, immune evasion, and therapeutic resistance. Targeting lactate metabolism, lactylation‐associated regulators, and immune checkpoint pathways may therefore offer promising opportunities for precision therapy in esophageal cancer.

1. Introduction

Esophageal cancer (EC) ranks as the seventh leading cause of cancer‐related deaths worldwide and is one of the deadliest cancers affecting the gastrointestinal system. From a histological perspective, EC is primarily divided into two principal types: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). ESCC predominantly occurs in the middle and upper segments of the esophagus and is more common in China and East Asia; EAC, on the other hand, predominantly occurs in the lower segment of the esophagus and is the predominant subtype in Europe and the United States [1, 2]. The main risk factors for esophageal cancer include dietary habits, lifestyle, and certain environmental exposures [3]. Despite advances in surgery, radiotherapy, chemotherapy, immunotherapy, and targeted therapy, the overall prognosis of patients with advanced esophageal cancer remains poor [4, 5]. Tumor progression, immune escape, and therapeutic resistance remain major obstacles to effective treatment. In normal cells, energy production under aerobic conditions mainly depends on mitochondrial oxidative phosphorylation (OXPHOS), whereas insufficient oxygen availability promotes a shift toward glycolysis. By contrast, tumor cells exhibit a distinct metabolic phenotype, preferentially using glycolysis for energy generation even in the presence of adequate oxygen, a phenomenon referred to as the Warburg effect [6, 7]. Abnormal lactic acid‐related metabolism drives tumor progression [8, 9]. Lactate metabolism, together with lactate‐induced disturbances in glucose and fatty acid metabolic pathways, outlines a metabolic evolutionary landscape within the tumor ecosystem, working in concert to drive tumor progression.

Traditionally, lactate has been viewed as a waste product or by‐product of metabolism [10, 11]. The identification of lactylation in 2019 paved the way for new research into the biological roles and molecular processes associated with lactate [12]. Lysine lactylation, a new type of post‐translational modification (PTM) mediated by lactate, can take place on histones as well as non‐histone proteins [13]. Lactylation of histones primarily contributes to changes in chromatin state and the regulation of gene transcription. Lactylation of non‐histones can regulate protein stability, interactions, subcellular localisation, and function. Regardless of the regulatory mechanism, both are closely engaged in various biological processes. The effects of lactate‐induced lactylation are mediated by several molecular mechanisms and signaling pathways, frequently working in conjunction with other epigenetic modifications to jointly enhance malignant characteristics [14, 15].

In esophageal cancer, enhanced glycolysis leads to increased glucose uptake, elevated lactate production, and acidification of the tumor microenvironment. Multiple molecules in esophageal cancer can promote glycolysis. Lactate is not only a metabolic waste product but also an important signaling molecule and substrate for epigenetic regulation. Lactylation may be a key mechanism linking metabolic abnormalities, transcriptional reprogramming, alterations in the immune microenvironment, and treatment resistance. This article systematically summarizes the mechanisms underlying the role of lactylation in the development and progression of esophageal cancer, following the central pathway of “metabolic reprogramming—lactic acid accumulation—lactylation—transcriptional/proteolytic remodeling—immune evasion and treatment resistance,” and discusses its potential clinical translational value as a diagnostic, prognostic, and therapeutic target.

2. The Biological Basis of Lactate Modification

2.1. Lactate Metabolism and the Lactate Shuttling System

Lactate plays a central role in regulating energy metabolism and signal transduction. It serves as an important cellular fuel, particularly under conditions in which oxygen supply and ATP demand are imbalanced, such as during intense exercise or infection. When cellular oxygen availability is restricted in hypoxic or anaerobic settings, it leads to a disruption in mitochondrial oxidative phosphorylation, which in turn causes inadequate utilization of pyruvate. During these circumstances, cells primarily depend on glycolysis to produce energy, eventually resulting in the formation of lactate. Lactate is primarily generated as a typical by‐product of glucose metabolism throughout the glycolytic pathway. In conditions of low oxygen or absence of it, glucose undergoes metabolism in the cytoplasm, resulting in the production of two pyruvate molecules [16]. However, when the electron transport chain is deprived of sufficient oxygen to act as the final electron acceptor, the mitochondria cannot efficiently oxidize pyruvate. In these situations, lactate dehydrogenase facilitates the transformation of pyruvate into lactate, all the while also regenerating NAD⁺, which is essential for the ongoing process of glycolysis.

Lactate metabolism in tumor cells differs markedly from that in normal cells; even under conditions of sufficient oxygen, tumor cells still rely primarily on glycolysis rather than mitochondrial oxidative phosphorylation to generate energy [17]. The Warburg effect supports the rapid proliferation of tumor cells in oxygen‐rich environments by relying primarily on glycolysis for energy; furthermore, it promotes tumor growth by providing essential growth factors and assists tumors in evading immune recognition [17]. Lactate metabolism and a brief overview of the lactylation process are shown in Figure 1.

FIGURE 1.

Lactic acid metabolism and the lactate shuttle. Schematic overview of lactate metabolism and lactylation. Glucose, glutamine, and extracellular lactate contribute to pyruvate, acetyl‐CoA, and lactyl‐CoA production, connecting glycolysis, the TCA cycle, and lipid/cholesterol synthesis. Lactate‐derived intermediates mediate histone and non‐histone lactylation through metabolic and signaling pathways. α‐KG, α‐ketoglutarate; ATP, adenosine triphosphate; CoA, coenzyme A; GLO‐1, glyoxalase 1; GLO‐2, glyoxalase 2; GPR81, G protein‐coupled receptor 81; GPR132, G protein‐coupled receptor 132; GSH, glutathione; LDH, lactate dehydrogenase; LGSH, lactoylglutathione/S‐D‐lactoylglutathione; MCTs, monocarboxylate transporters; MGO, methylglyoxal; PDH, pyruvate dehydrogenase; TCA cycle, tricarboxylic acid cycle.

Lactate enters the cell through four reversible monocarboxylate transporters (MCTs), such as MCT1 and MCT4 [18, 19]. The expression of MCT1, which is regulated by c‐Myc, is widespread across different cell types, facilitating the transport of both lactate and pyruvate. In contrast, MCT4 is a highly effective transporter for lactate, activated under hypoxic conditions, and is predominantly found in glycolytic tissues like fast‐twitch muscle fibers and various cancer cells [20, 21]. Many solid tumors depend significantly on glycolysis for their energy needs. Increased expression of MCTs in tumor cells fosters the development of an acidic microenvironment, which is crucial for the survival of cancer cells. Evidence suggests that lactate transport‐related pathways also exhibit abnormal expression and functional significance in esophageal cancer. Studies have shown that MCT1 and MCT4 are expressed in esophageal adenocarcinoma cells; AZD3965, a selective MCT1 inhibitor, increases intracellular lactate accumulation and reduces cell viability, suggesting that MCT‐mediated lactate efflux may be a potential therapeutic target [22]. In esophageal squamous cell carcinoma, the expression of MCT1, MCT4, CD147, and GLUT1 is associated with tumor pathological characteristics; specifically, MCT1 is associated with higher TNM staging and metastasis, whilst MCT4 and CAIX positivity are indicative of poorer overall survival. Functional experiments have also demonstrated that the combined inhibition of MCT1 and MCT4 reduces ESCC cell viability and extracellular lactate accumulation [23]. Lactate shuttling serves three main physiological roles: it acts primarily as an energy source, functions as a precursor for gluconeogenesis, and plays a role as a signaling molecule exhibiting autocrine, paracrine, and endocrine‐like activities [24, 25]. Lactate produced by muscles can be converted into glucose through gluconeogenesis in the liver and subsequently transported back to the muscles, a process classically described as the Cori cycle. MCTs mediate the intercellular transport of lactate to target cells. Current research has shown that lactate shuttling contributes to interactions among different cell populations within the tumor microenvironment (TME). This form of lactate exchange has been defined as “metabolic symbiosis” and represents a key phenomenon in tumor biology [24, 26].

Lactate plays a role in signal transduction mechanisms within cancer cells. In particular, lactate acts to inhibit prolyl hydroxylases that are dependent on 2‐oxoglutarate (PHDs), with a focus on PHD2. This inhibition prevents the ubiquitination and subsequent proteasomal degradation of hypoxia‐inducible factor‐1α (HIF‐1α), which is facilitated by the tumor suppressor protein Von Hippel–Lindau (VHL), thereby contributing to the stabilization of HIF‐1α [27]. Furthermore, lactate can inhibit the PHD2/VHL system via a pathway that is independent of HIF‐1 but acts synergistically with the HIF‐1 pathway, thereby inducing angiogenesis and maintaining tumor metabolism under hypoxic conditions. In addition to acting as an intracellular signaling mediator, lactate can also function as an extracellular ligand [28]. G protein‐coupled receptor 81 (GPR81) is a specific G protein‐coupled receptor for lactate; by binding to GPR81, lactate supports energy metabolism in tumor cells. In clinical pancreatic cancer samples, 148 out of 158 patients (94%) exhibited high GPR81 expression. Functionally, knocking down GPR81 under culture conditions containing lactate and low glucose resulted in a significant decrease in mitochondrial activity and induced extensive pancreatic cancer cell death [29].

In addition to GPR81, the G protein‐coupled receptor G2A (GPR132) is also a key transmembrane lactate receptor, similarly mediating immunosuppression and metastasis [30, 31]. Lactate activates GPR132 on the surface of macrophages, promoting their polarization toward the M2 phenotype and enhancing the adhesion, migration, and invasive capabilities of breast and lung cancer cells [32].

2.2. Lactylation: A Bridge Linking Metabolism and Epigenetic Regulation

Histone lactylation commonly occurs at lysine residues. It tends to accumulate in gene promoter or enhancer regions, where it promotes the transcription of specific genes by altering chromatin accessibility. In esophageal cancer, histone lactylation promotes gene expression. Crucially, lactylation alters not only histones to modulate gene transcription but also affects non‐histone proteins, thus impacting various cellular functions such as gene expression, enzyme functionality, protein stability, subcellular localization, protein interactions, and other types of post‐translational modifications [33, 34]. Multiple studies indicate that in response to stimuli like hypoxia, interferon‐γ (IFN‐γ), lipopolysaccharide (LPS), or bacterial infections, histone lactylation levels in the cytoplasm change (Kla) increase and localize to the genome, thereby promoting lactate production [35]. Histone Kla has been reported in various cancers, particularly the lactylation of histone H3 at lysine 18 (H3K18la) [36, 37]. H3K18la has been shown to participate in various biological processes, including tumourigenesis, tumor progression, tumor immune evasion, and metabolic reprogramming in cancer cells [38]. Elevated expression of N⁶‐methyladenosine RNA‐binding protein 2 (YTHDF2) in ocular melanoma cells has been attributed to increased Kla levels in its promoter region. Furthermore, as an m⁶A reader, YTHDF2 recognizes m⁶A‐modified mRNAs encoding the circadian rhythm regulator PER1 and the tumor suppressor p53 (TP53), promoting their degradation and thereby accelerating ocular melanoma progression [36]. Furthermore, Kla is also involved in chemotherapy resistance. Lactate plays a key role in DNA repair and chemotherapy resistance through lactylation modification by the NBS1 protein; lactate can significantly enhance homologous recombination repair (HRR). After tumor cells undergo DNA damage caused by chemotherapy, lactate can rapidly repair the damaged DNA, thereby reducing the therapeutic effect and leading to the development of resistance [39].

Regarding non‐histone lactylation, one proposed mechanism is a non‐enzymatic pathway that utilizes lactyl‐glutathione (LGSH) as a substrate. Methylglyoxal (MGO), which is generated as a by‐product of glycolysis, interacts with glutathione via the enzyme thioesterase glyoxalase I (GLO1) to produce LGSH. This compound is then converted back into glutathione and D‐lactate through hydrolysis by GLO2 [40]. Additional research has confirmed its involvement in facilitating tumor advancement. Alanine‐tRNA synthetase 1 (AARS1) has the capacity to detect lactate and initiate site‐specific lactylation of p53, resulting in a reduction of p53's ability to bind DNA and fostering liquid–liquid phase separation (LLPS), thus diminishing the tumor‐suppressive role of p53 in a mouse model of colorectal cancer (CRC) [41]. AARS2 has also been identified as a regulator of cellular metabolism via lactylation by limiting the entry of acetyl‐CoA during the processes of pyruvate and fatty acid oxidation, which ultimately hampers oxidative phosphorylation [42, 43]. Collectively, these insights have significantly enhanced our comprehension of non‐histone lactylation. Lactic acid is capable of inducing lactylation (Kla) of methyltransferase‐like protein 16 (METTL16) at the K229 residue, which subsequently facilitates the m⁶A modification of ferredoxin 1 (FDX1) mRNA, boosts FDX1 mRNA levels, and leads to cell death in gastric cancer through the cuproptosis pathway [44]. Moreover, akin to histone lactylation, modifications in non‐histone lactylation are also implicated in resistance to tumor therapies. The lactylation of the non‐histone protein meiotic recombination 11 homolog 1 (MRE11) improves the capacity for homologous recombination (HR) repair and is associated with chemoresistance in colorectal cancer [45].

Current research trends indicate that histone and non‐histone lactylation are developing in parallel, with the latter receiving increasing attention. A growing body of evidence highlights the critical role of lactylation in various diseases, particularly in tumors characterized by elevated lactate levels. Overall, existing evidence has elucidated multiple mechanisms of non‐histone lactylation regulation: firstly, metabolic regulation, whereby metabolic enzymes are directly modified to influence their activity and stability; secondly, transcriptional regulation, whereby gene expression is modulated through the lactylation of transcription factors; and thirdly, structural regulation, whereby the cellular framework is influenced through the modification of structural proteins.

2.3. The “Write‐Erase‐Read” Mechanism of Lactylation

The regulation of lactylation by enzymatic activity involves distinct enzymes known as “writers,” “erasers,” and “readers.” Lactyltransferases, referred to as the “writers,” play a crucial role in attaching a lactyl group to lysine residues. This group of enzymes includes P300/CBP, GCN5, TIP60, AARS1‐2, and KAT8. In contrast, the enzymes that remove the lactyl group, known as de‐lactyltransferases or the “erasers,” comprise HDAC1‐3, 8 and SIRT1‐3 (which are deacetylases 1–3) [46]. Notably, SIRT3 is capable of inhibiting the development of esophageal squamous cell carcinoma by modulating gene transcription through specific changes in H3K9la [47]. Future studies are needed to further investigate the mechanisms that underlie lactylation.

“Readers” are responsible for recognizing lactylation sites. Although no specific lactylation readers have yet been identified, their potential role in the lactylation process cannot be overlooked. Lactylation is not a passive accumulation of lactate‐derived marks; rather, it is characterized by reversibility and enzymatic regulation. Long‐standing questions regarding the biosynthesis of lactyl‐CoA have recently been addressed, with studies confirming that the enzyme pairs ACSS2/KAT2A and GTPSCS/p300 are responsible for adding lactyl groups to lysine residues [48].

2.4. Cross‐Talk Between Lactylation and Other Post‐Translational Modifications

Lysine acetylation is a thoroughly characterized post‐translational modification (PTM) that was initially discovered on histones in 1963. The potential functional overlap between lactylation and acetylation remains a significant question for ongoing research. Commonalities between the two modifications stem from the fact that both lactate and acetyl‐CoA originate primarily from pyruvate, the final product of glycolysis, and their molecular frameworks bear resemblance [49, 50]. Both modifications employ p300 as a catalytic “writer” for lysine, while class I–III histone deacetylases (HDAC1–3) function as “erasers” [51, 52]. Nevertheless, a proteomics investigation of Kupffer cells revealed 2198 acetylation sites and 289 lactylation sites, with only 46 sites showing overlap. Additionally, the motif characteristics (i.e., unique sequence patterns) surrounding these two types of modifications exhibited no similarities [53].

Studies on the lactylation and acetylation of the same protein site suggest that these two modifications may have distinct functions or may be in a competitive relationship. Zong et al. found that the lactylation of p53 at the K120 and K139 positions hinders acetylation at these sites and disrupts both the capacity for DNA binding and the liquid–liquid phase separation (LLPS) of p53, consequently preventing its activation [41]. Lactylation mediated by HDAC6 primarily affects soluble tubulin dimers instead of the microtubules directly, whereas the acetylation of α‐tubulin at the K40 position takes place within the lumen of the microtubule. As a result, lactylation and acetylation do not engage in direct competition on the microtubules [54]. Overall, these studies suggest that lactylation and acetylation are regulated by distinct mechanisms, potentially arising from the synergistic action of “writing enzymes,” “erasing enzymes” and metabolic factors. Even when occurring at the same protein site, lactylation and acetylation may perform different functions and exhibit subtle interactions under specific conditions.

3. Lactate Metabolism and Lactylation in Esophageal Cancer

In recent years, numerous studies have reported on lactylation processes associated with esophageal cancer, highlighting their potential clinical applications as biomarkers and therapeutic targets. It remains unclear how the specific lactylation patterns in esophageal cancer differ from those in other tumors. A diagram illustrating the mechanism of action of lactylation in esophageal cancer is shown in Figure 2. The signaling pathways involved in lactylation are listed in Table 1.

FIGURE 2.

Lactate signaling pathways: Hypoxia, immune evasion and copper‐mediated cell death. This schematic depicts lactylation‐mediated crosstalk in esophageal cancer. Hypoxia‐induced glycolysis increases lactate, promoting histone lactylation and transcriptional programs that enhance proliferation, migration, invasion, stemness, and glycolysis. Lactate also suppresses CD8+ T‐cell function and regulates PLAU, NUD21, and FDX1‐associated pathways, contributing to immunosuppression and cuproptosis resistance within the tumor microenvironment and progression. AARS1, alanyl‐tRNA synthetase 1; CD8+ T cell, cluster of differentiation 8‐positive T cell; GPRC5A, G protein‐coupled receptor class C group 5 member A; GZMB, granzyme B; HDAC2, histone deacetylase 2; HIF‐1α, hypoxia‐inducible factor 1‐alpha; H3K18la, histone H3 lysine 18 lactylation; H3K9la, histone H3 lysine 9 lactylation; H4K12la, histone H4 lysine 12 lactylation; IFN‐γ, interferon‐gamma; lac, lactylation; LAMC2, laminin subunit gamma 2; LOXL2, lysyl oxidase‐like 2; MTHFD1L, methylenetetrahydrofolate dehydrogenase 1‐like; NIPAL1, NIPA‐like domain‐containing 1; PLAU, plasminogen activator urokinase; Pol II, RNA polymerase II; p300, E1A binding protein p300; SHMT2, serine hydroxymethyltransferase 2; TNF‐α, tumor necrosis factor‐alpha.

TABLE 1.

Signaling pathways involved in lactate modification in esophageal cancer.

Modification type	Upstream factor	Molecule/site	Downstream target/pathway	Biological process involved in esophageal cancer
Histone lactylation	Hypoxia/lactate	H3K9la	LAMC2	Invasion [55]
Histone lactylation	NIPAL1‐HCK‐LDHA/p300	H3K18la	NIPAL1	Immune escape [56]
Histone lactylation	PDIA3P1‐GLUT1/HK2	H4K8la	BMP7	Progression [57]
Histone lactylation	EVA1A‐lactate	H4K12la	PLAU	CD8+ T cell suppression [58]
Histone variant lactylation	Lactate	H2BC9 K44la	Wnt7b/Wnt/β‐catenin	Proliferation/drug resistance [59]
Non‐histone lactylation	Lactate/AARS1	NUDT21 K23la	FDX1 APA	Cuproptosis resistance [60]
Non‐histone lactylation	Hypoxia	HIF‐1α K172la	HIF‐1 complex	Immune escape [61]
Non‐histone lactylation	Hypoxia	Axin1 K147la	Axin1 ubiquitination	Glycolysis/stemness [62]
Non‐histone lactylation	Hypoxia	SHMT2 lactylation	MTHFD1L	Glycolysis/progression [63]

3.1. Lactylation and Esophageal Cancer Cell Proliferation

It has been shown that the lactylation of specific histones as well as non‐histone proteins can affect the proliferation of diverse tumor cells. The circular RNA circXRN2 engages the Hippo pathway by stabilizing LATS1, which subsequently inhibits tumor progression associated with H3K18 lactylation in tumors [64]. Additionally, research has indicated that AARS1 is upregulated and moves into the nucleus, where it directly facilitates the lactylation of YAP (at the K90 position) and TEAD1 (at the K108 position), thus enhancing the expression of downstream target genes and furthering the proliferation of gastric cancer cells [65].

Furthermore, PDIA3P1 disrupts the interaction between membrane‐associated RING‐CH8 (MARCH8) and hexokinase 2 (HK2), which diminishes HK2's ubiquitination and degradation, thereby fostering glycolysis. Active glycolysis results in significant lactate production, leading to an increase in histone H4K8 lactylation (H4K8la) levels and stimulating the transcription of the target gene bone morphogenetic protein 7 (BMP7). This highlights the critical function of the PDIA3P1‐modulated glycolysis–H4K8la–BMP7 signaling pathway in the advancement of ESCC [57]. Knockdown of AP001885.4 attenuates histone lactic acid modification and NF‐κB (p65) protein expression; conversely, lactic acid modification inhibitors (2‐DG, 2‐deoxy‐D‐glucose and oxalamide) and NF‐κB inhibitors (JSH‐23) also reduce c‐myc expression [66]. The H2B variant H2BC9 is an oncogene associated with lactylation; lactylation at the H2BC9 K44 site activates Wnt7b transcription and the downstream Wnt/β‐catenin signaling pathway, thereby driving the progression of ESCC [59].

3.2. Lactylation and Hypoxia

Glycolysis is significantly enhanced under hypoxic conditions, primarily manifested by increased glucose uptake, enhanced lactate production, and increased expression and activity of key metabolic enzymes [67]. Hypoxia‐enhanced glycolysis promotes the progression of esophageal cancer. Therefore, inhibiting hypoxia‐induced glycolysis in esophageal cancer may represent a promising strategy for suppressing the progression of the disease [68, 69].

Hypoxia treatment enhances glycolysis and cellular stemness in esophageal cancer cells while raising the lactylation levels of the Axin1 protein. The lactylation induced by hypoxia at K147 of the Axin1 protein facilitates its ubiquitination, which in turn boosts glycolysis and cellular stemness in TE1 and EC109 cells [62]. In esophageal cancer tissues and cells, SHMT2 shows significant upregulation; hypoxia elevates SHMT2 protein levels, consequently fostering the proliferation, migration, invasion, stemness, and glycolysis of EC cells. The lactylation of the SHMT2 protein induced by hypoxia increases its protein levels, leading to an upregulation of MTHFD1L expression and promoting the malignant progression of esophageal cancer cells [63]. Research indicates that hypoxia stimulates histone H3K9Lac, which enhances the transcription of LAMC2, thereby facilitating the proliferation of ESCC. Histone H3K9Lac encourages LAMC2 expression, which subsequently contributes to the invasion of ESCC cells [55].

3.3. The Role of Lactylation in Cell Death

Lactylation of proteins in tumor cells is linked to tumor cell death, with modifications in lactylation triggering various cell death pathways, such as pyroptosis, ferroptosis, and copper‐mediated cell death. A recent investigation demonstrated a notable functional relationship between the lactylation of HDAC1, RNA demethylation, and the resistance to ferroptosis in colorectal cancer (CRC) [70].

The lactylation process of NUDT21 increases its binding affinity with CPSF6, facilitates the formation of the CFIm complex, and leads to the elongation of the 3′ untranslated region (UTR) of FDX1. This elongation results in a decrease in protein production, consequently providing resistance to copper‐mediated cell death in esophageal squamous cell carcinoma (ESCC). AARS1 serves as the “writer” that catalyzes the lactylation at the K23 site of NUDT21, while HDAC2 plays the role of the enzymatic “eraser.” Increased levels of LDHA and NUDT21, along with enhanced lactylation of NUDT21 at the K23 site, correlate with lower FDX1 expression and a worse prognosis for patients with ESCC [60].

3.4. Lactylation Modification and Immune Evasion

Immune evasion plays a crucial role in tumourigenesis. Notably, various studies have established a link between protein lactylation and reduced immune recognition of malignant cells [71, 72]. Research indicates that lactylation affecting both histones and non‐histones can facilitate immune evasion in tumor cells. Within the tumor microenvironment (TME), histone lactylation aids in creating an immunosuppressive setting: it influences the transcription of downstream genes related to DNA damage repair (such as Arg1 and Klf4) and promotes the polarization of M2 macrophages [73, 74]. In the case of tumor‐specific cytotoxic T lymphocytes (CTLs), histone lactylation enhances the transcription of circATXN7. This elevation of circATXN7 subsequently binds to the NF‐κB p65 subunit, obscuring its nuclear localization signal motif and keeping it in the cytoplasm, which increases CTLs' susceptibility to activation‐induced cell death, consequently resulting in immune evasion by tumor cells [75].

Research has indicated that a novel prognostic model involving three prognostic genes (NONO, H2BC18, POLDIP3) is associated with immune infiltration. This model demonstrated excellent predictive accuracy in both internal and external datasets [76]. Another study identified a NIPAL1‐driven metabolic‐epigenetic circuit associated with esophageal squamous cell carcinoma (ESCC) that promotes the growth of tumors while simultaneously inhibiting anti‐tumor immunity. The levels of expression of NIPAL1, phosphorylated LDHA (Y10), and H3K18la correlate with the effectiveness of immune checkpoint blockade therapy [56]. Increased EVA1A expression facilitates glycolysis in ESCC; this leads to a rise in lactate that further triggers lactylation of H4K12, which in turn enhances PLAU expression, thereby undermining the anti‐tumor responses of CD8+ T cells [58]. Hypoxia plays a critical role in immune evasion within esophageal cancer; research indicates that combining HIF‐1α inhibition (PX‐478) with anti‐PD‐1 therapy yields a synergistic effect, dramatically improving tumor management and boosting the cytotoxic function of CD8 T cells. This investigation demonstrates that lactylation at the K172 site of HIF‐1α is fundamental to the hypoxia‐mediated immune evasion observed in ESCC [61]. It is evident that the lactylation process plays a crucial role in anti‐tumor immunity against esophageal cancer. Furthermore, given its close association with hypoxia, this targeted therapy holds significant therapeutic promise.

4. Lactylation Modification and the Immune Microenvironment

4.1. Lactate Accumulation and the Formation of an Immunosuppressive TME

Lactylation may serve as a critical hub within the metabolic–epigenetic interactions that influence the immunosuppressive tumor microenvironment (TME), as illustrated in Figure 3. Gaining a comprehensive understanding of the intricate nature of the TME and the relationships between its various cell types is essential for advancing precision cancer treatment. Due to the distinctive and complex features of the TME, it includes numerous fibroblast subpopulations, a poorly developed vascular network, and a wide variety of immune cells, primarily characterized by immunosuppressive components [77, 78]. Among other cell populations, the interaction between lactate and immune cells can lead to impaired cell differentiation, attenuated immune responses, and immune evasion, resulting in compromised immune surveillance and reduced responsiveness to treatment [79].

FIGURE 3.

Lactylation and the tumor immune microenvironment. Schematic representation of lactylation‐mediated immune regulation in the tumor microenvironment. Lactylation promotes immunosuppressive phenotypes in Tregs, T cells, NK cells, dendritic cells, B cells, and tumor‐associated macrophages, enhancing immune evasion, altered metabolism, apoptosis resistance, and M2‐like macrophage polarization during tumor progression. Bregs, regulatory B cells; CTLA‐4, cytotoxic T‐lymphocyte‐associated protein 4; DCs, dendritic cells; GLUT10, glucose transporter 10; IFN‐α, interferon‐alpha; IFN‐γ, interferon‐gamma; IL‐2, interleukin‐2; IL‐6, interleukin‐6; lac, lactylation; MCT11, monocarboxylate transporter 11; M2/M1, M2‐like/M1‐like macrophage phenotype ratio; NFAT, nuclear factor of activated T cells; NK cells, natural killer cells; OXPHOS, oxidative phosphorylation; PD‐L1, programmed death‐ligand 1; PD‐1, programmed cell death protein 1; TAMs, tumor‐associated macrophages; TNF, tumor necrosis factor; Tregs, regulatory T cells.

4.2. Lactate Modulation and Tumor‐Associated Macrophages

Lactylation links metabolic reprogramming with immune epigenetic regulation by modulating METTL3, PD‐L1, and macrophage inhibitory factor (MIF) that are involved in facilitating immune evasion in colorectal cancer (CRC). Within macrophage‐related pathways, proprotein convertase subtilisin/kexin type 9 (PCSK9), a crucial molecule for cholesterol metabolism, is found to be significantly elevated in CRC [80]. Silencing PCSK9 leads to a decrease in histone lactylation and MIF expression, while fostering anti‐tumor M1 polarization and inhibiting pro‐tumor M2 macrophages, thus hampering the progression and metastasis of CRC [81]. The presence of lactate and histone lactylation encourages the transformation of macrophages into M2‐type tumor‐associated macrophages (TAMs); these TAMs subsequently amplify the expression of M2‐associated genes, including hypoxia‐inducible factor 2α (HIF‐2α), arginase 1 (ARG1), and vascular endothelial growth factor (VEGF).

Although much of the current evidence regarding the regulation of tumor‐associated macrophages by lactylation derives from other tumors, these findings provide important insights for research into esophageal cancer. Given that esophageal cancer is characterized by marked glycolytic activity, lactate accumulation and an immunosuppressive microenvironment, future research should focus on validating the spatial distribution of lactylation markers, lactate transporters and macrophage subsets in samples from esophageal cancer patients. Concurrently, by combining single‐cell sequencing, spatial transcriptomics, proteomics, patient‐derived organoids, co‐culture systems, and in vivo models, it will be possible to further clarify whether lactylation is involved in the formation of immunosuppressive macrophages in squamous cell carcinoma and adenocarcinoma of the esophagus and to assess whether targeting lactate metabolism or lactylation regulatory factors can improve the efficacy of immunotherapy for esophageal cancer.

4.3. Lactate Modulation and T‐Cell Dysfunction

With regard to regulatory T cells (Tregs), a lactate‐rich microenvironment promotes Treg proliferation and enhances their immunosuppressive effects [82, 83]. In a TME characterized by high glycolysis, lactate can elevate levels of programmed cell death ligand 1 (PD‐L1) on Tregs, leading to reduced efficacy of immunotherapy [84, 85]. It has been established that Treg cells infiltrating tumors need lactate uptake to preserve their immunosuppressive capabilities. Research focusing on Treg‐specific MCT1 knockout has revealed that while lactate is not critical for the functioning of peripheral Treg cells, it plays an essential role within the tumor microenvironment (TME); its absence may impede tumor progression and improve the responsiveness to immunotherapy [86].

In melanoma lacking LDHA, there is a notable increase in the presence of NK cells and CD8+ cytotoxic T cells. Liu et al. reported that lactate generated by KRAS‐mutant colorectal cancer cells can diminish the effectiveness of anti‐PD‐1 therapy by deactivating nuclear factor κB (NF‐κB) and increasing CD8+ T cells' susceptibility to activation‐induced cell death (AICD) [87, 88].

4.4. Lactate Modulation and NK Cells

Natural killer (NK) cells are a key cytotoxic effector component of the innate immune system, capable of rapidly eliminating transformed or infected cells and stimulating immune responses. As they rely on oxidative phosphorylation (OXPHOS) rather than glycolysis to maintain survival and function, NK cells are particularly vulnerable in lactate‐rich microenvironments. Elevated lactate in the TME can directly impair the cytotoxic activity of NK cells and induce apoptosis, whilst simultaneously attenuating NKT cell function by inhibiting the mTOR pathway and blocking the nuclear translocation of the promyelocytic leukemia zinc finger protein (PLZF). These effects are partly mediated by NFAT inhibition, leading to reduced production of IFN‐γ and IL‐4 [89, 90].

5. Lactylation‐Related Biomarkers and Potential Therapeutic Targets in Esophageal Cancer

Current research suggests that lactylation‐related modifications are involved in esophageal cancer cell proliferation, glycolysis, hypoxic adaptation, stem cell maintenance, immune evasion, and treatment resistance, and may serve as potential biomarkers for prognostic assessment and treatment response prediction. Lactylation‐related biomarkers in esophageal cancer mainly comprise three categories: firstly, lactylation‐related prognostic genes or feature models, such as the risk scoring model comprising NONO, H2BC18, and POLDIP3, which can predict the prognosis and immune infiltration status of ESCA patients; secondly, histone acetylation markers, such as H3K18la, H4K12la, H3K9la, H4K8la, and H2BC9 K44la; third, non‐histone lactylation markers, such as HIF‐1α K172la, Axin1 K147la, SHMT2 lactylation, and NUDT21 K23la, which promote tumor progression by regulating transcriptional complex assembly, protein stability, glycolysis, and resistance to apoptosis and copper‐mediated cell death.

5.1. p300 Inhibitors

p300/CBP is an epigenetic regulator with histone acetyltransferase activity; in addition to mediating acetylation, it also acts as a “lactylase,” utilizing lactyl‐CoA to promote histone lysine lactylation. p300/CBP inhibitors include catalytic inhibitors, bromo‐domain inhibitors, and other small‐molecule inhibitors; among these, C646 and A‐485 directly inhibit its enzymatic activity. In ESCC, NIPAL1 recruits HCK and promotes phosphorylation of LDHA at Y10, thereby enhancing glycolysis and lactate production; lactate further induces p300‐mediated H3K18la, activating NIPAL1 transcription and forming a “NIPAL1‐HCK‐p‐LDHA‐lactate‐p300‐H3K18la” positive feedback loop. p300 inhibitors can block this loop, reduce H3K18ac levels, restore CD8⁺ T‐cell function, and enhance the efficacy of PD‐1 antibodies; they are suitable for ESCC patients with high NIPAL1 expression or active H3K18ac [56].

5.2. HCK Inhibitors

HCK is a non‐receptor tyrosine kinase of the Src family involved in the regulation of inflammation, immune signaling, and tumor metabolism. HCK inhibitors include Src family kinase inhibitors such as dasatinib and bosutinib, as well as more selective small‐molecule drugs. In ESCC, HCK, upon recruitment by NIPAL1, phosphorylates LDHA at position Y10, thereby enhancing LDHA activity, promoting lactate accumulation, and the formation of H3K18la. Inhibition of HCK blocks LDHA phosphorylation and lactate production at an upstream level, disrupting the NIPAL1–H3K18la positive feedback loop and reducing lactate‐dependent immune evasion. Studies suggest that HCK inhibition restores the effector function of CD8⁺ T cells and enhances sensitivity to PD‐1 antibody therapy; therefore, HCK is a key target linking tyrosine kinase signaling, lactate metabolism, and histone lactylation [56].

5.3. HIF‐1α Inhibitors

HIF‐1α is a central transcription factor in the tumor cell response to hypoxia, participating in enhanced glycolysis, angiogenesis, and immune evasion. PX‐478 is a commonly used HIF‐1α inhibitor that reduces HIF‐1α expression and the transcription of its downstream target genes. In ESCC, hypoxia induces protein lactylation and promotes the assembly of the HIF‐1 transcriptional complex. Lactylation at the HIF‐1α K172 site is crucial for its binding to HIF‐1β and the activation of target genes, driving immunosuppressive programmes such as glycolysis, TGF‐β, and Notch. PX‐478 inhibits hypoxic adaptation and, in synergy with anti‐PD‐1 therapy, enhances CD8⁺ T‐cell infiltration and the expression of granzyme B, IFN‐γ, and TNF‐α; it is suitable for ESCC subgroups with significant hypoxia or elevated HIF‐1α K172la [61].

5.4. The Glycolysis Inhibitor 2‐DG

2‐Deoxy‐D‐glucose (2‐DG) is a glucose analogue that competitively inhibits glucose metabolism, reducing glycolytic flux, ATP production, and lactate generation. As lactate is an important substrate for lactic acid modification, 2‐DG indirectly reduces the levels of histone and non‐histone lactylation. In esophageal cancer, hypoxia induces Axin1 K147 lactylation, promoting the ubiquitination and degradation of Axin1, thereby enhancing glycolysis and cellular stemness; Axin1 mutants can further enhance the inhibitory effects of 2‐DG on glycolysis and stemness [62]. Therefore, 2‐DG is suitable for esophageal cancer characterized by active glycolysis, increased lactate production, and elevated H3/H4 lactylation levels.

5.5. LDHA Inhibitors

LDHA is a key enzyme that catalyzes the conversion of pyruvate to lactate and is essential for maintaining glycolysis and lactate accumulation. LDHA inhibitors include FX11, GNE‐140, threonine analogues, and stevopentol, which possesses LDHA inhibitory activity. In ESCC, LDHA serves both as a lactate‐producing enzyme and as an upstream target for lactate‐based therapy. The NIPAL1‐HCK axis promotes LDHA Y10 phosphorylation, inducing lactate production and H3K18a; lactate also induces NUDT21 K23 lactylation, promoting FDX1 3′UTR elongation, reducing FDX1 protein expression and leading to copper‐mediated resistance to cell death. Steripentol reduces lactate production, attenuates NUDT21 lactication, and synergistically inhibits tumor growth with the copper ion transporter elesclomol [60].

5.6. Anti‐PD‐1 Antibodies

PD‐1 is an immune checkpoint receptor on the surface of T cells; anti‐PD‐1 antibodies can block PD‐1/PD‐L1 signaling and restore the antitumour function of T cells. Lactylation modifications may influence the response to immunotherapy in esophageal cancer. The NIPAL1‐HCK‐LDHA‐H3K18la loop can impair CD8⁺ T cell function via lactate accumulation and p300‐mediated histone lactylation; while HIF‐1α K172la promotes hypoxia‐induced immunosuppressive transcriptional programmes [56]. Inhibition of HCK, p300, or HIF‐1α can all enhance the efficacy of PD‐1 antibodies. Therefore, NIPAL1, p‐LDHA Y10, H3K18la, and HIF‐1α K172la may serve as potential biomarkers for selecting patients for combination immunotherapy.

5.7. Copper Ion Carrier Elesclomol

Elesclomol is a copper ion carrier that facilitates copper entry into cells and induces copper‐mediated cell death. In ESCC, lactate induces lactylation of NUDT21 at K23, enhancing its interaction with CPSF6, promoting FDX1 3′UTR elongation, and reducing FDX1 protein output, thereby conferring resistance to copper‐mediated cell death. Combining the LDHA inhibitor stevulopentol with elesclomol reduces lactate production and NUDT21 lactylation whilst restoring FDX1 expression; simultaneously, it enhances copper‐dependent cytotoxic effects, synergistically inhibiting tumor growth [60]. Consequently, the combination of “LDHA inhibitor + copper ion carrier” represents a potential therapeutic strategy based on the regulation of copper‐mediated cell death sensitivity via lactylation.

5.8. HDAC2 Inhibitors

HDAC2 belongs to the class I histone deacetylase family, and some studies suggest it may also function as a de‐lactylase. HDAC2 inhibitors include broad‐spectrum HDAC inhibitors such as vorinostat, panobinostat, and romidepsin. In studies on NUDT21 lactylation, HDAC2 has been identified as the “eraser” of NUDT21 K23la. Consequently, inhibiting HDAC2 may increase NUDT21 lactylation, promote FDX1 3′UTR elongation, and enhance resistance to copper‐induced cell death, posing a potential risk of promoting drug resistance [60]. For ESCC patients with high NUDT21 K23la expression and low FDX1 expression, non‐selective HDAC2 inhibitors should be used with caution, or a reverse strategy to enhance HDAC2 lactylation activity should be considered.

5.9. SIRT3 Activators

SIRT3 is an NAD⁺‐dependent deacetylase that primarily maintains mitochondrial metabolic homeostasis. Recent studies indicate that SIRT3 acts as a H3K9la‐specific deacetylase, regulating the transcription of related genes. SIRT3 activators or strategies to enhance SIRT3 expression, such as NAD⁺ precursor supplementation and metabolic modulators, may reduce abnormal acetylation levels. In ESCC, hypoxia enhances H3K9la and promotes LAMC2 transcription, driving tumor invasion; conversely, SIRT3 can inhibit tumor progression by removing H3K9la. Therefore, enhancing SIRT3 function may help block the H3K9la‐LAMC2 axis, and SIRT3 expression and H3K9la levels may also serve as biomarkers for assessing lactate‐dependent progression [47].

6. Future Research and Outlook

As research into tumor metabolic reprogramming continues to advance, lactylation—as a key link between abnormal glucose metabolism and epigenetic regulation—is gradually emerging as a new area of focus in esophageal cancer research. Current evidence suggests that lactylation not only contributes to cell proliferation, invasion, maintenance of stemness, immune evasion, and treatment resistance in esophageal cancer, but also influences the tumor microenvironment and treatment response by regulating the function of histones and non‐histones. However, current research remains predominantly focused on basic and mechanistic exploration, and there is still considerable ground to cover before clinical translation. Although lactylation is involved in tumor progression, immune evasion, and treatment resistance in a variety of tumors, its role in esophageal cancer has yet to be systematically elucidated. Esophageal cancer is characterized by significant metabolic reprogramming, a hypoxic microenvironment, chronic inflammation, and resistance to radiotherapy and chemotherapy, all of which are closely associated with lactate accumulation and the regulation of lactylation. It is therefore necessary to specifically investigate the potential role of lactylation in esophageal cancer. The relevant mechanisms currently require further validation in esophageal cancer models and clinical samples. Moving forward, the first priority is to further refine the lactylation profile of esophageal cancer, systematically identifying key lactylation sites, regulatory enzymes, and effector molecules across different histological subtypes, stages, and treatment contexts, and clarifying their spatiotemporal roles in tumourigenesis and progression. Secondly, clinical validation of lactylation‐related biomarkers should be strengthened. This involves advancing association analyses between candidate markers—such as H3K18la, H3K9la, HIF‐1α K172la, and NUDT21 K23la—and patient prognosis, immune infiltration status, and drug sensitivity, thereby establishing a testing system for risk stratification and treatment response prediction.

Furthermore, the development of more specific targeted drugs centred on the “lactate production—lactylation epigenetic modification—downstream signaling pathways” represents a key future direction. In addition to classical targets such as p300, LDHA, and HIF‐1α, SIRT3, HDAC2, and non‐histone lactylation regulatory networks also warrant further investigation. Concurrently, attention should be paid to optimizing combination therapy strategies, such as the synergistic use of lactylation‐targeting drugs with immune checkpoint inhibitors, radiotherapy, and chemotherapy, metabolic therapies, and copper‐mediated cell death inducers, with the aim of improving treatment outcomes for esophageal cancer, particularly squamous cell carcinoma of the esophagus. Overall, research into lactylation is expected to provide new biological evidence and intervention targets for the precision diagnosis and treatment of esophageal cancer.

Analyses based on public genomic databases and clinical samples can provide important external validation for research into lactate‐mediated modifications in esophageal cancer. TCGA data show that molecules such as EVA1A and PLAU are abnormally expressed in esophageal squamous cell carcinoma and are correlated with genes related to glycolysis and lactate metabolism, such as LDHA, PKM, and SLC2A1, suggesting that enhanced lactate production may be involved in the progression of esophageal cancer and the remodeling of the immune microenvironment. Furthermore, the abnormal upregulation of candidate molecules such as NIPAL1 in clinical ESCC tissues, and their association with disease staging and prognosis, further supports the clinical significance of lactate metabolism‐related pathways in patient samples. Although this evidence cannot yet directly prove a causal role for lactate modification, it serves as an important complement to in vitro experimental results, providing a basis for subsequent lactate detection, functional validation, and mechanistic studies in patient tissues.

In summary, lactylation plays a significant role in the development and progression of esophageal cancer, involving multiple processes such as metabolic reprogramming, epigenetic regulation, immune evasion, and treatment resistance. The relevant biomarkers and targets possess potential value for diagnosis, prognostic assessment, and therapeutic applications, and are expected to provide new insights for the precision treatment of esophageal cancer.

Author Contributions

Xiaowei Liu: conceptualized and wrote the manuscript. Weizhuang Sun: prepared Figures 1, 2, 3. Changyong Li and Yang Wang: provided guidance and significant advice about the manuscript.

Funding

The authors have nothing to report.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

No new data.