The moonlighting functions of glycolytic enzymes in tumorigenesis

Abstract

Glycolysis is a ubiquitous energy-supply process that occurs in virtually all living cells. The phenomenon of aerobic glycolysis, known as the Warburg effect, is observed and considered as a hallmark of tumorigenesis. However, the roles and unresolved mechanisms of glycolytic enzymes in cancer progression remain incompletely understood. In this review, we synthesize emerging knowledge on the non-glycolytic (“moonlighting”) functions of key glycolytic enzymes in tumorigenesis, which are independent of their canonical catalytic activity and enable them to govern diverse cellular processes beyond energy metabolism. We also discuss their potential as therapeutic targets and establish a conceptual framework for understanding how these moonlighting activities govern tumor progression, thereby supporting the development of novel anticancer therapies.

Clinical trial number

Not applicable.

Introduction

Glycolysis is a universal glucose metabolic process present in all living organisms. It involves a series of enzymatic reactions that split glucose into two pyruvate molecules while extracting energy [1, 2]. A hallmark of cancer cells is their increased glucose metabolism and reprogramming towards aerobic glycolysis [3]. This metabolic adaptation enables tumor cells to fulfill their heightened energy demands [4]. Interestingly, a similar metabolic shift is also observed under inflammatory conditions [5]. Nevertheless, the roles, the mechanistic insights and unresolved questions surrounding critical glycolytic enzymes in tumorigenesis remain inadequately emphasized. In this review, we summarize the non-canonical functions of glycolytic enzymes in tumor, which extend beyond their traditional roles in glycolysis. We also highlight the functions and outstanding challenges related to these enzymes in tumor progression and evaluate promising therapeutic strategies for cancer treatment.

Elevated glycolysis is a hallmark of tumor cells

In the 1920s, Otto Warburg observed that tumor cells displayed enhanced glucose uptake and produced large amounts of lactate, even in the presence of adequate oxygen conditions with functional mitochondria—a phenomenon now known as the “Warburg effect” [6, 7]. In human cells, glycolysis begins with the import of glucose into the cytoplasm by glucose transporters (GLUTs). Notably, GLUT1 plays a pivotal role in coordinating this process during tumor metabolic reprogramming. Due to its role in enhancing tumor glycolysis dependence and maintaining treatment resistance, which contributes to the progression of malignancy, it has also attracted the attention of researchers [8]. The rate and direction of glycolysis is regulated by multiple cytoplasmic enzymes. These enzymes include hexokinase (HK), glucose-6-phosphate isomerase (GPI), phosphofructokinase (PFK), aldolase, Triosephosphate isomerase 1 (TPI1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGAM), enolase, and pyruvate kinase (PK) [9] as shown in Fig. 1. Among them, the key rate-limiting steps involve the conversions catalyzed by HK (glucose to glucose-6-phosphate), PFK (fructose-6-phosphate to fructose-1, 6-phosphate), and PK (phosphoenolpyruvate to pyruvate). It has been reported that monocarboxylate transporter 4 (MCT4)-mediated lactate export represents a critical regulatory node in the glycolytic pathway. In summary, four major rate-limiting steps control glycolytic flux in tumors [10]. Additionally, lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate, playing a critical role in regulating glycolytic flux [11] (Fig. 1).

Fig. 1.

Overview of glycolytic pathways. The rate-limiting enzymes are marked in red, and non-rate-limiting enzymes are marked in blue

The nonclassical functions of rate-limiting glycolytic enzymes in tumorigenesis

Growing evidence show that rate-limiting glycolytic enzymes such as HKs, PFKs PKMs, and MCT4 in tumorigenesis extends to non-metabolic functions, whereby their moonlighting activities promote proliferation, metastasis, and drug resistance. These effects are mediated through the activation of key pathways including Wnt/β-catenin, as well as through immune and epigenetic mechanisms.

HKs promote tumor development and predict poor prognosis

The HK family, including HK1, HK2, HK3, glucokinase (GCK), and hexokinase domain-containing 1 (HKDC1), catalyzes the phosphorylation of glucose to glucose-6-phosphate (G6P), committing glucose to the glycolytic pathway [12, 13]. This canonical function is essential for maintaining glycolytic flux and energy production in both normal and tumor cells. Among the isoforms, HK1-3 isoforms have a high glucose affinity; GCK exhibits lower affinity [14]. HKDC1 displays relatively low enzymatic activity [15]. Functioning as a housekeeping gene [16], HK1 is ubiquitously expressed across mammalian tissues [14] and shows minimal expression changes under physiological stresses [17]. In contrast, HK2 shows high embryonic tissue expression and responds to various hormonal or metabolic stimuli [14, 17]. Whereas, HK3 is widely but minimally expressed in tissues. GCK serves as a glucose sensor in pancreatic, hepatic, cerebral and intestinal tissues [18]. HKDC1 exhibits tissue-specific expression [15].

Although multiple HK isoforms are implicated in tumor growth, metastasis [19–21] and poor prognosis [22], HK2 emerges as the most active isozyme [23] and is aberrantly overexpressed in most aggressive tumors [24]. Moreover, HK2 is required for tumor initiation and exerts distinct oncogenic functions through its dynamic subcellular localization [12, 25], positioning it as a promising therapeutic target in cancer.

The roles and unsolved mysteries of subcellular HK2 in tumorigenesis

Accumulating studies demonstrate that hexokinase 2 (HK2) localizes to the cytoplasm, mitochondria, and nucleus, exerting distinct, location-dependent functions in tumors. In the cytoplasm, HK2 enhances glycolytic flux to boost metabolic activity and drive tumor malignancy. Beyond its canonical role, cytoplasmic HK2 interacts with CD133 and recruits the deubiquitinase ubiquitin-specific protease 11 (USP11), stabilizing CD133 and promoting cancer stemness—a mechanism linked to tumor recurrence and therapy resistance [26]. Furthermore, under high glucose conditions, HK2 dissociates from mitochondria, binds to and phosphorylates IκBα at T291 in human glioma cells, leading to PD-L1 upregulation, which in turn promotes tumor immune evasion [27]. HK2-driven glycolysis in tumor pericytes also supports tumor angiogenesis [28]. In human ovarian cancer, HK2 activates Wnt/β-catenin pathway to upregulate Cyclin D1/c- Myc, accelerating cell proliferation and tumor formation [29].

In mitochondria, HK2 localization is regulated by SUMOylation at K315 and K492 [16]; it exhibits context-dependent roles: promoting pyroptosis in head and neck cancer [30], while inhibiting tumorigenesis in other settings [16]. Mitochondrial HK2 binds BAX, inhibiting BAX-induced cytochrome C release and apoptosis [31]. Moreover, in hepatocellular carcinoma (HCC) cells, mitochondrial HK2 confers resistance to natural killer (NK) cell-mediated cytolysis [32].

In the nucleus, HK2 functions independently of its kinase activity to drive stemness and chemoresistance of acute myeloid leukemia (AML) by enhancing chromatin accessibility and maintaining DNA integrity [33]. Meanwhile, HK2 further influences the tumor immune microenvironment by modulating CD8⁺ T cells and Treg dynamics, fostering an immunosuppressive/pre-tumor microenvironment formation [34]. These multifaceted roles underscore HK2 as a central regulator of tumorigenesis and cancer progression (Fig. 2).

Fig. 2.

HK2 drives tumorigenesis via multiple pathways. HK2 promotes the stemness and proliferation of tumor cells in the cytoplasm, and promotes tumor angiogenesis in pericytes. Mitochondrial HK2 affects pyroptosis, cytolysis, and promotes tumor immune escape. The nuclear HK2 promotes tumor stemness and chemoresitance

The advantages of HK2 inhibitors are evident in tumor treatment

Given that chemotherapy resistance poses a significant obstacle in cancer treatment, and considering multifaceted role of HK2 drives tumor development, HK2 has emerged as a valuable therapeutic target. Genetic deletion of HK2 suppresses tumor initiation [12] and sensitizes HCC cells to metformin [35]. We previously demonstrated that silencing HK2 with shRNA also inhibits glioma (GM) proliferation in vivo and in vitro [7]. Targeting HK2 reverses cisplatin resistance in ovarian cancer [36, 37]. Combining HK2 inhibitors (2-Deoxyglucose (2-DG), arsenic trioxide (ATO), Gen-27, etc) with chloroquine (CQ), an inhibitor of ULK1-dependent autophagy, achieves near-complete tumor suppression [38]. In addition, limonin disrupts mitochondrial HK2 binding and sensitizes HCC cells to mitochondrial apoptosis [39]. The miR-143/145 tumor-suppressor cluster inhibits renal cell carcinoma (RCC) cell proliferation and invasion by downregulating HK2 [40].

Numerous HK2-targeting agents have shown promise across tumor types (Table 1). Benitrobenrazide blocks tumor growth by inhibiting HK2-mediated glycolysis [68]. 2-DG prohibits proliferation and potentiates the anti-tumor effects of chemotherapy compounds such as metformin and sorafenib [69, 70]. Metformin disrupts HK1/2 activity to inhibit proliferation [41]. The pyruvate analog 3-bromopyruvate (3-BrPA) synergizes with daunorubicin through direct HK2 inhibition [71]. Lonidamine alone or in combination with doxorubicin has advanced to clinical trials [72–75]. Additionally, a variety of natural compounds including ATO and astragalin, exert anti-tumor effects via HK2 suppression [47, 76]. Collectively, these findings underscore HK2 as a compelling target for cancer treatment. Clinical evaluation of HK2 inhibitors may expedite their translation into therapies, particularly for early-stage malignancies (Table 2).

Table 1.

Pharmaceutical drugs targeting glycolytic enzymes in tumor treatment

Enzyme	Agent	Phase	Description	Ref	Toxicity
HK2	2-deoxy-d-glucose	II	Targets glucose binding site	[41]	Metabolic disorders (Long-term or high-dose)
	3-BP	Preclinical	Inhibits the interaction of HK2 with VDAC1	[42]	hepatotoxicity/nephrotoxicity at high doses
	Gen-27	Preclinical	Enhances the antitumor effect of sorafenib	[43]	Unclear
	Shikonin	Preclinical	Inhibits cell viability	[44]	Embryonic developmental toxicity and vasodilatory effect
	Ikarugamycin	Preclinical	Affects glycolysis pathway	[45]	Unclear
	Lonidamine	III/II	Combination therapy	[46]	Unclear
	Arsenic trioxide	I/II	Induces apoptosis of cancer cells	[47]	Hepatotoxicity
	GL-V9	Preclinical	Induces mitochondrial cytotoxicity	[41]	Unclear
	Chrysin	Preclinical	Reduces HK2 expression	[48]	Genetic toxicity
PFKFB3/PFK1	3-BP	Preclinical	Inhibits glycolytic activity	[49]	/
	PFK15	Preclinical	Induces apoptosis, cell cycle arrest	[50]	Unclear
	PFK158	I	Induces apoptosis	[51]	Unclear
PKM2 Inhibitors	Shikonin	Preclinical	Induces cell cycle arrest	[52]	/
	Curcumin	Preclinical	Inhibits glucose uptake and Lactate production	[53]	Liver toxicity at High-dose
	Resveratrol	Preclinical	Induces apoptosis	[54]	Reduce blood glucose at High-dose
	Proanthocyanidin B2	Preclinical	Induces apoptosis	[55]	Unclear
	Benserazide	Preclinical	Reduces cell proliferation	[56]	Mild liver toxicity
PKM2 Activators	PA-12	Preclinical	Blocks PKM2 nuclear localization	[57]	Unclear
	ZINC08383544	Preclinical	Affects the expression of genes mediated PKM2 during glycolysis	[58]	Unclear
	Compound 0089–0022	Preclinical	Induces apoptosis	[59]	Unclear
PGAM1	MJE3	Preclinical	Covalent labelling	[60]	Unclear
	EGCG	II	Affects glycolysis	[61]	Unclear
	PGMI-004A	Preclinical	Prohibits cell proliferation	[62]	Unclear
Enolase 1	ENOblock	Preclinical	Enhances anti-tumor immunity in combination with immunotherapy	[63]	Unclear
LDHA	Berberine	Preclinical	Functional inhibitors	[64]	Anti-blood coagulation
	FX11	Preclinical	Reduces tumor growth	[65]	Unclear
	Gossypol	I/II	Induces cell cycle arrest	[66]	Fluctuations
ALDOA	UM0112176	Preclinical	Induces apoptosis	[67]	Unclear

Abbreviations: 2-DG, 2-deoxy-d-glucose; 3-BP, 3-Bromo-2-oxopropanoic acid ethyl ester

Table 2.

Comparison of the transformation potential of glycolytic enzymes

Enzyme	Clinical translational potential	Inhibitors	Challenge	Cancers
HK2	2-DG, Lonidamine and Arsenic trioxide have been proven effective in the phase II clinical trials of combined treatment. Arsenic trioxide increases the efficiency of conventional chemotherapy drugs by 30% in neuroblastoma. Other drugs are in the preclinical and have potential.	2-DG, 3-BP, Gen-27, Shikonin, Ikarugamycin, lonidamine, Arsenic trioxide, GL-V9, Chrysin	On-target toxicity (HK2 expression in cardiac and neural tissues) insufficient specificity (for example, 2-DG also targets HK1).	Liver cancer, glioma, ovarian cancer, lung cancer, etc.
PFKFB3 /PFK1	In the Phase I clinical trial of PFK158, the safety dosing assessment for patients has been completed. The Phase II clinical trial has not yet been completed. 3-BP and PFK15 is in the preclinical.	3-BP, PFK15, PFK158	Low oral bioavailability and the specificity	Gastric cancer, lung cancer, breast cancer, chronic myeloid leukemia
PKM2	Moderate potential. Inhibitors such as Shikonin, Curcumin, Resveratrol and Benserazide are still in the preclinical stage and the experiments are still ongoing.	Shikonin, Curcumin, Resveratrol, Benserazide	Metabolic plasticity (such as switching to oxidative phosphorylation) can easily lead to drug resistance. The complex regulatory mechanisms.	Lung cancer, colorectal cancer, leukemia
LDHA	Gossypol showed a complete response in 11 of 13 patients in the phase II clinical trial for gastric esophageal cancer, with a median progression-free survival of 52 months. Berberine and F×11 in the preclinical.	Berberine, FX11, Gossypol	Tend to trigger compensatory mechanisms (such as the upregulation of LDHB).	Solid tumors

The glycolytic functions of PFKs in tumor development

PFK catalyzes the third key rate-limiting step of glycolysis, converting fructose-6-phosphate and ATP into fructose-1,6-bisphosphate—a critical commitment step that significantly enhances glycolytic flux. As a bifunctional enzyme, PFK serves as a critical intermediate regulator of glycolysis. There are two different PFK enzymes in humans [77]. Phosphofructokinase 1 (PFK1) catalyzes the conversion of fructose 6-phosphate and ATP into fructose-1,6-diphosphate and ADP, representing one of the most essential control points governing glycolytic rate [78, 79]. On the contrary, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/PFKFB), which harbors the highest kinase activity among its four bifunctional isoenzymes, generates fructose 2,6-bisphosphate (F-2,6-BP) from fructose 6-phosphate, leading to PFK1 activation.

PFK1 consists three tissue-specific isoforms: phosphofructokinase platelet (PFKP), phosphofructokinase muscle (PFKM) and phosphofructokinase liver (PFKL). Under physiological conditions, PFKP and PFKM are expressed in platelets and muscle, respectively. PFKL is the dominant isoform in liver and kidney [80]. The PFKFB enzyme family, responsible for both the synthesis and degradation of F-2,6-BP [81], includes four members: PFKFB1, PFKFB2, PFKFB3, and PFKFB4 [81].

Beyond the established roles of PFK2 in cardiac glycolysis and diabetic heart disease [82], the functions of PFK1 and PFK2 have been extensively investigated in tumor development [83–85].

The moonlighting functions and challenges of PFKs in tumor development

Overexpression of these three PFK1 isoforms has been reported in a variety of cancer types including breast cancer and ovarian cancer [83–85]. Each isoform promotes proliferation, cell survival and metastasis, and could serve as a biomarker for poor prognosis in tumor patients [86–89].

Up-regulation of PFKP increases glycolysis and lactate production, which in turn stimulates cancer cell migration and metastasis [83, 90], the resulting lactate-rich acidic tumor microenvironment further supports tumorigenesis. Beyond promoting the Warburg effect, PFKP engages in crosstalk with oncogenic signaling pathways such as epidermal growth factor receptor (EGFR). On one hand, activated EGFR acetylates PFKP at K395, promoting its translocation to the plasma membrane, where it is phosphorylated at Y64 by EGFR. This modification triggers AKT activation via a PI3K-dependent mechanism, which subsequently reinforces PFKP activity through PFK2-mediated phosphorylation, thereby driving tumor growth [91]. On the other hand, PFKP facilitates EGFR-induced nuclear translocation and activation of β-catenin, thereby enhancing expression of Cyclin D1 and Myc [92]. PFKP stabilization by activated AKT1 is also essential in glioblastoma (GBM) [85]. In addition, PFKP stabilization mediated by activated AKT1 is required for in GBM [85]. Notably, miR-520 directly targets PFKP and suppresses HCC progression [93]. ZEB1-induced PFKM transcription promotes HCC carcinogenesis and metastasis [94]. Elevated expression of PFKM is linked to epithelial ovarian cancer susceptibility [95] and confers bevacizumab resistance in GBM [96]. We have found that AKT2-regulated PFKM stabilization is essential for anti-tumor macrophage-mediated immunity [97]. Our unpublished data further demonstrate that PFKM is shuttled into nucleus by binding p53, potentiating p53 transcriptional ability to induce PD-1 expression, which impairs macrophage phagocytosis. In addition, S-nitrosylation of PFKM stabilizes its tetrameric form and promotes tumor progression [98]. Polymorphisms in PFKM also affect epithelial ovarian cancer susceptibility in Han women from Southwest China [87]. Similarly, high PFKL activity correlates with invasiveness in ovarian cancer [99], and PFKL induces cancer cell proliferation by governing lipolysis and enhancing β-oxidation [100] (Fig. 3).

Fig. 3.

The role of PFK isoenzymes in tumor development. PFK isoenzymes regulate tumor processes such as metastasis and chemoresistance via multiple pathways

Recent studies reveal the roles of PFKFB3 and PFKFB4 in tumor progression such as breast cancer, colorectal cancer (CRC) and pancreatic cancer [101–103]. Both enzymes promote proliferation, invasion, and drug-resistance, and their elevated expression correlates with shorter overall survival (OS) in several malignancies [104]. In lung cancer cells, fascin promotes tumor growth and metastasis by increasing PFKFB3 expression [105]. PFKFB3 overexpression also promotes angiogenesis and metastasis in breast cancer by increasing the expression of vascular endothelial growth factor-α (VEGF-α) [106]. In chronic myeloid leukemia (CML), PU0.1-induced PFKFB3 elevation associates with imatinib-resistance [107]. Nuclear PFKFB3 regulates cyclin-dependent kinase (CDKs) activity and inhibits cell death in HCC [108, 109]. Meanwhile, PFKFB4 enhances cell proliferation by phosphorylating and activating transcriptional activity of SRC-3 in breast cancer [110], and promotes chemoresistance with poor survival in small-cell lung cancer (SCLC) patients [104], highlighting PFKFBs importance in tumorigenesis (Fig. 3).

The promising treatment strategy of targeting PFKs in vivo

Pharmacological PFK inhibition demonstrates therapeutic benefits. Several PFK1 inhibitors have been reported (Table 1). Clotrimazole (CTZ) suppresses glycolysis by inhibiting PFK1 [111]. Aspirin, a widely used anti-inflammatory agent, exerts dose-dependent anti-tumor effects by altering glucose metabolism and inducing apoptosis. Aspirin could directly attenuate glycolysis through inhibition of PFK [112]. Combined treatment with aspirin and sorafenib overcomes sorafenib resistance and induces HCC apoptosis via PFKFB3 inhibition [113]. Given PFKFB3’s role in PFK1 activation, PFKFB inhibitors have attracted research interest. Silencing or pharmacologically inhibiting PFKFB3 reduces AKT activation, impairs DNA repair, and induces cell cycle arrest [114, 115]. In CML, PFKFB3 inhibition suppresses cell growth and significantly prolongs survival of both allograft and xenograft mouse mice [107]. PFK15, a small molecule PFKFB3 inhibitor, exerts anti-glycolytic and anti-tumor effects in gastric cancer (GC) by inducing apoptosis and cell cycle arrest [50]. PFK158, a new PFKFB3 inhibitor, targets the active form of PFKFB3^ser461, attenuates cancer stemness, and induces apoptosis in SCLC cells enriched cancer stem cells (CSC) [116]. PFK-158 undergoes in phase I clinical trials (Table 2) [117]. In addition, 5MPN, a selective inhibitor PFKFB4, suppresses CD44-induced tumorigenesis [118]. Although progress has been made, current glycolytic inhibitors have limited efficacy against PFK1. Therefore, the development of direct PFK1 inhibitors is urgently needed.

PK isoforms expression and functions

PK catalyzes the final rate-limiting step of glycolysis, converting phosphoenolpyruvate and ADP into pyruvate and ATP [119, 120]. This irreversible reaction is essential for directing glycolytic carbon flux and maintaining energy homeostasis in both normal and malignant cells. Mammals express four PK isoforms—PKM1, PKM2, PKL and PKR—each with distinct tissue distributions and regulatory properties [121]. PKM1 is expressed in myocardium, skeletal muscle, and brain tissue. PKM2 predominates in proliferative cells such as during embryonic development and in tumors [122]. PKL is expressed in liver, kidney, and erythrocytes [123]. PKR is erythrocyte-specific [123]. Unlike other tetrameric PK isoforms, PKM2 exists in both tetrameric and dimeric conformations, which exhibit differential biological activities [124]. Post-translational modifications, including phosphorylation and acetylation, govern PKM2‘s subcellular localization and non-glycolytic functions, thereby contributing to tumor progression [125, 126].

PKM2 drives tumorigensis through its non-glycolytic functions

Most cancers preferentially express PKM2 over PKM1 [127, 128]. Although PKM1 activates glucose catabolism, stimulates autophagy/mitophagy, and favors malignancy in certain contexts such as SCLC [129], PKM2 remains the dominant oncogenic isoform. Silencing PKM2 and restoring PKM1 expression reverses the Warburg effect and suppresses tumor growth in human cancer cell lines [130], demonstrating that PKM2-to-PKM1 switching inhibits tumorigenesis [130–132]. Collectively, these studies establish PKM2‘s essential role in promoting tumor progression (Fig. 4).

Fig. 4.

PKM2 affects the development of tumors. PKM2 promotes tumor growth by maintaining its dimeric conformation, which upregulates VEGFA and phosphorylates Bcl-2. Conversion to the tetrameric form reverses these effects, inhibiting tumorigenesis

Beyond its glycolytic role, PKM2 drives tumor development through promoting angiogenesis, inhibiting apoptosis, inducing multidrug resistance, promoting stemness and metastasis, and altering the inflammatory response. In contrast to its tetrameric form in normal tissues, in tumor cells, PKM2 predominantly exists as a dimer, and promotes tumorigenesis by multiple mechanisms [133, 134]. Serine binding activates PKM2 and stimulates cell proliferation [135]. The RNA export factor ALYREF stabilizes PKM2 mRNA by binding to m5C sites in its 3’-untranslated regions, enhancing glycolysis in bladder cancer cells [136]. Protein arginine deiminase 1 (PADI1) and PADI3 citrullinate PKM2; re-programming its ligand interactions to promote hyper-glycolysis and proliferation [137]. O-GlcNAcylation of PKM2, mediated by O-GlcNAcase, increases aerobic glycolysis and tumor growth [138]. Upon hypoxic stress, PKM2 partners with NF-κB for nuclear import. This complex subsequently promotes VEGFA expression through direct recruitment to its promoter; additionally, PKM2-HIF-1α binding enhances HIF-1α accumulation and VEGF-α secretion to promote angiogenesis [139, 140]. Pharmacological inhibition of PKM2 reduces angiogenesis [141]. Under oxidative stress, PKM2 translocates to the mitochondria in GM cells, where it phosphorylates Bcl-2 at Thr69, driving an anti-apoptotic effect [125]. PKM2 also suppresses apoptosis by inhibiting the caspase-8/caspase-3/GSDME signaling [142].

Nuclear PKM2 induces gefitinib resistance via STAT3 activation [143], and NOX4 stabilizes PKM2 protein to confer drug resistance [144]. Under stress conditions, nuclear PKM2 binds to Oct4 and activates stemness-related genes [145]. Separately, in bladder cancer, DPYSL2 facilitates PKM2 dimerization, a process that drives malignant progression by boosting glycolysis and inducing epithelial-mesenchymal transition [145]. Moreover, the oxidation of methionine residue (M239) maintains PKM2 in a tetrameric state to promote metastasis. Paradoxically pharmacological activation of PKM2 by TEPP46 increases pancreatic cancer metastasis in vivo [146]. PKM2 also modulates the tumor immune microenvironment. HCC-secreted PKM2 promotes the differentiation of monocytes into pro-tumor macrophages, facilitating tumor development [147, 148]. These observations strengthens PKM2‘s multifaceted role in tumor progression.

Although numerous studies implicate PKM2 in tumorigenesis, its essential role has been challenged. In a Brca1-deficient mouse model of breast cancer, deletion of PKM2 unexpectedly accelerates mammary tumor formation, indicating that PKM2 is dispensable for tumor development, while total pyruvate kinase (PK) activity remains necessary in quiescent tumor cells [132]. Tumor cells appear capable of modulating PKs’ activity according to metabolic demands [149]. The discrepancies highlight the need to clarify the context-dependent roles and necessity of PKM2 in future studies.

Targeting PKM2 conformation as a therapeutic strategy

Modulation of PKM2 conformation represents a promising therapeutic avenue for cancer patients. Clinically, PKM2 phosphorylation has been identified as a potential target in triple-negative breast cancer [150]. PKM2 expression serves as a prognostic marker in renal cancer [151]. Current PKM2-targeting strategies follow two main approaches. One aims to inhibit the dimeric form to block its nuclear translocation and associated oncogenic functions, while the other seeks to promote tetramerization to restore normal glycolytic flux. As summarized in Table 1, several PKM2-targeting inhibitors and activators have shown efficacy across multiple cancer types, demonstrating the therapeutic potential of manipulating the dimer-tetramer equilibrium of PKM2 in anticancer treatment.

MCT4 exerts its effects in glycolysis and tumors by regulating lactate

The lactate transport mediated by MCT4 plays a crucial role in maintaining the intracellular pH level and the stability of lactate, plays a controlling role in the flow of glycolysis [10]. MCT4 plays a critical role in tumor progression by facilitating lactate efflux. It is highly expressed in hypoxic regions of tumors under the regulation of HIF-1α, where it helps maintain intracellular pH homeostasis, thereby enabling cancer cells to sustain high glycolytic rates essential for rapid proliferation [152]. Beyond its metabolic support, MCT4 significantly shapes the tumor microenvironment: exported lactate acidifies the extracellular space, inhibiting the function of CD8⁺ T cells and NK cells while promoting the activity of immunosuppressive cells such as Treg cells and myeloid-derived suppressor cells [153]. This lactate-rich milieu also facilitates epithelial-mesenchymal transition, angiogenesis, and metastasis [152]. Moreover, Moreover, elevated MCT4 expression is associated with poor prognosis in various cancers, including triple-negative breast cancer (TNBC) and GBM [152].

Therapeutically, inhibiting MCT4 disrupts lactate export, causing intracellular acidification and suppressing glycolytic flux, thereby selectively imposing metabolic stress on tumor cells [152]. Combining MCT4 inhibitors with immune checkpoint blockers may alleviate immunosuppression and enhance antitumor immunity, while dual inhibition of MCT4 and MCT1 (a lactate importer) can disrupt metabolic symbiosis between tumor cells. Syrosingopine have shown preclinical efficacy in impairing tumor growth through MCT4 inhibition [154]. Thus, targeting MCT4 offers a compelling strategy to counteract metabolic adaptation and immune evasion in aggressive cancers.

The roles of non-rate-limiting enzymes in tumor development

A number of studies have revealed that non-rate-limiting enzymes contribute to tumor progression by orchestrating key processes such as proliferation, metastasis, drug resistance, angiogenesis and immune evasion. Their expression and activity levels thus represent valuable prognostic predictor in various cancers (Fig. 5).

Fig. 5.

The role of non-rate-limiting enzymes in regulating tumor growth by a variety of ways. Non-rate-limiting enzymes regulate tumor proliferation, resistance, metastasis, immune response and angiogenesis, and could be poor predictor for cancer patients

GPI expression negatively correlates with cancer prognosis

GPI is a member of the glucose phosphate isomerase family. It catalyzes the reversible isomerization between D-glucose-6-phosphate and D-fructose-6-phosphate, thereby playing a central role in both glycolysis and gluconeogenesis [155]. GPI plays a significant role in tumor progression, mediated by its cytokine and growth factor activity [156]. Indeed, GPI promotes cancer metastasis [157–159], although the underlying mechanisms remain incompletely elucidated. Clinically, elevated GPI expression is negatively correlated with prognosis in breast cancer [160], GC [158] and clear cell-renal cell carcinoma [161]. Recent studies indicate that GPI expression is inversely associated with infiltrated CD8⁺ T cells, and its inhibition sensitizes tumor cells to CD8⁺ T cell-mediated killing [162, 163]. Bestatin, a clinically advanced aminopeptidase inhibitor, attenuates immune evasion in ovarian cancer by blocking GPI attachment and reducing CD24 expression on tumor cells [164].

Aldolase influences tumor progression via enzymatic and non-enzymatic functions

Positioned midway in glycolysis, aldolase catalyzes the reversible cleavage of fructose-1,6-bisphosphate into dihydroxyacetone-3-phosphate and glyceraldehyde-3-phosphate. Among its three isoforms: muscle isoform (ALDOA), ALDOB, and ALDOC, ALDOA is the predominant isoform in most cancers, and serves as an independent prognostic marker in cancers like HCC [165] and CRC [166]. Beyond glycolysis, ALDOA drives HCC progression by promoting mRNA translation and protein synthesis independent of its catalytic activity [167]. ALDOA also interacts with HIF-1 to promote epithelial-mesenchymal transition in CRC [167]. Inhibition of ALDOA by UM0112176 induces cancer cell apoptosis by disrupting the actin cytoskeleton and mitochondrial homeostasis [67]. ALDOB exhibits context-dependent roles: its aberrant expression promotes metastatic growth in HCC [168], whereas hepatic ALDOB deficiency activates AKT signaling and accelerates HCC development [169]. Conversely, ALDOB suppresses HCC via inhibiting glucose-6-phosphate dehydrogenase in a p53-dependent manner [170].

The oncogenic role of TPI1 could rely on its subcellular localization

TPI1 converts the reversible interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. TPI1 is upregulated and promotes metastasis in GC and breast cancer [171, 172], serving as a potential predictor of poor prognosis in breast cancer [172], lung adenocarcinoma (LUAD) [173] and laryngeal squamous cell carcinoma (LSCC) [174], though it is opposite in HCC patients [175]. Mechanistically, TPI1 stabilizes cell division cycle associated 5 (CDCA5), activating the PI3K/AKT/mTOR pathway to drive breast cancer progression [172]. Moreover, nuclear-localized TPI1 contributes to LUAD development and chemoresistance [176]. Although several inhibitors targeting TPI1’s catalytic site have been identified [177], their clinical relevance in oncology remains unexplored, underscoring the need for further investigation into TPI1’s functional roles.

Post-translational modifications of GAPDH critically regulate tumorigenesis

GAPDH specifically catalyzes the reversible conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. Beyond glycolysis, GAPDH controls DNA repair, autophagy, carcinogenesis, and cell death [178–180]. Its overexpression is correlated with poor prognosis [181, 182]. Post-translational modifications affect GAPDH localization and function [183]. AKT2-mediated phosphorylation blocks GAPDH nuclear translocation, attenuating apoptosis of ovarian cancer cells [184], while reduced S-nitrosylation retains cytoplasmic GAPDH in the cytoplasm and suppresses nuclear-mediated apoptosis [185]. ADP-ribosylation, carbonylation and acetylation also regulate its apoptosis function [186–188]. GAPDH interacts with Sp1 to induce Snail expression, promoting epithelial-mesenchymal transition and metastasis [189]. In T cells, GAPDH overexpression drives angioimmunoblastic T-cell lymphoma via NF-κB activation [190]. Conversely, recent study reveals that GAPDH Q262 serotonylation enhances glycolytic metabolism and CD8⁺ T cell anti-tumor activity [191]. GAPDH drives immune evasion and HCC development by upregulating PD-L1 expression [192]. Koningic acid (KA), a classical GAPDH inhibitor, lacks substantial evidence for clinical application in oncology, warranting further research.

The dual role of PGK in tumor progression

There are two isoforms of PGK, PGK1 and PGK2. In contrast to the ubiquitously expressed PGK1, PGK2 expression is restricted to spermatogenic cells [193, 194]. PGK1 is overexpressed in diverse cancers [195–198] and regulates proliferation [199], angiogenesis [200], epithelial-mesenchymal transition [201–203], autophagy [204] and mitochondrial metabolism [205]. High PGK1 expression predicts poor prognosis [206]. O-GlcNAcylation at threonine 255 (T255) enhances PGK1 activity, accelerating CRC growth [195]. High intracellular PGK1 expression promotes proliferation and drug resistance via AKT and β-catenin pathways [207–209], whereas high extracellular PGK1 suppresses tumor growth and angiogenesis [200]. PGK1 also modulates autophagy in response to oxygen levels [210]. Despite its oncogenic roles, few small-molecule inhibitors targeting PGK1 have been developed, highlighting a critical gap in therapeutic targeting.

PGAM promotes tumorigenesis via non-metabolic networks

PGAM catalyzes the interconversion of 3-phosphoglycerate and 2-phosphoglycerate. The PGAM family includes five isoforms (PGAM1-5), with PGAM1 (brain isoform) and PGAM2 (muscle isoform) being the most characterized [211]. Nuclear PGAM1 promotes migration independent of its metabolic activity [212]. High PGAM1/4 or nuclear PGAM2 correlates with poor prognosis [213–215], while the roles of PGAM3 and PGAM5 remain unclear. Loss of TP53 frequently upregulates PGAM1, increasing glycolysis and biosynthesis [216]. Silencing or inhibition of PGAM1 by shRNA or the molecule inhibitor PGMI-004A suppresses cell proliferation and tumor growth [62]. In addition, PGAM1 inhibition also potentiates anti-PD-1 immunotherapy by promoting ferroptosis and CD8⁺ T cell infiltration [217]. Under oncogenic RAS signaling, PGAM2 interacts with Chk1 to enhance glycolysis [218], and its inhibition overcomes enzalutamide resistance in metastatic prostate cancer [219]. PGAM4 knockdown impairs the GM cell growth, though the underlying mechanisms are unknown [215]. Given its pro-tumorigenic role, PGAM1 is a potential therapeutic target. Several PGAM1 inhibitors, such as MJE3, PGMI-004A, and EGCG (currently in Phase II trials, NCT06398405), are under evaluation (Table 1).

Multifaceted roles of enolases in oncogenesis

Enolase catalyzes the conversion of 2-phosphoglycerate into phosphoenolpyruvate. Mammals express three isoforms: α-enolase (ENO1), β-enolase (ENO3), and γ-enolase (ENO2). Enolases, especial ENO1 overexpression is observed in various cancers, and is associated with chemotherapy resistance and poor prognosis [220–233]. ENO1 could enhance the stemness of cancer stem cells by promoting glycolysis [234], and acts as an RNA-binding protein to facilitate YAP1 translation, triggering HCC progression through alternative arachidonic acid metabolism [235]. Additionally, ENO1 promotes choline metabolism by blocking CHKα degradation, accelerating cancer cell proliferation [236]. Dynamic O-GlcNAcylation of ENO1 modulates aerobic glycolysis and immune evasion [237]. An ENO1 DNA vaccine inhibits tumor growth and potentiates the anti-tumor ability of gemcitabine in pancreatic cancer models [238–240]. The ENO1 inhibitor ENOblock enhances anti-multiple myeloma immunity when combined with anti-PD-L1 therapy in preclinical studies [63], supporting its potential as a therapeutic target.

LDH as a therapeutic target in cancer

LDH comprises subunits LDHA (M) and LDHB (H) encoded by LDHA and LDHB respectively. LDH is assembled into five different combinations by various combinations of LDHA and LDHB subunits: LDH1 (4 H), LDH2 (3H1M), LDH3 (2H2M), LDH4 (1H3M) and LDH5 (4 M) [241]. LDHA predominates in skeletal muscle and liver, converting pyruvate to lactate. LDHB is predominantly expressed in the heart, brain, spleen, kidney, and erythrocytes, where it catalyzes the reduction of pyruvate to lactate [242]. Besides, LDHA and LDHB, LDHC and LDHD genes have been found in vertebrates [242, 243]. LDHC is a testis-specific gene [242]. LDHD is predominantly expressed in kidney and liver [243].

Elevated serum LDH levels correlates with poor prognosis across cancers [244, 245]. Hypoxia induces LDH activity in pancreatic tumor cells, producing L-2HG to regulate the stemness and immune evasion [246]. High levels of LDHA expression and phosphorylation are associated with poor prognosis in cancer patients [247–250]. Some factors induce tumor progression by modulating the post-translational modification of LDHA. CPT1A-mediated succinylation [251] and protein arginine methyltransferase 3-mediated arginine methylation of LDHA [252] promotes tumor progression. LDHA drives oncogenesis via multiple pathways. LDHA-recruited tumor-associated macrophages in GBM [253]. LDHA drives oncogenesis via Rac1 activation in a glycolysis-independent manner, promoting breast cancer development [254]. LDHA also promotes tumor progression by activating AKT pathway, TGF-β-activated kinase 1 (TAK1)/NF-κB signaling pathway, or genes transcription [255–257].

Similarly, LDHB is also aberrantly expressed in tumors and could be prognostic biomarker [258–260]. Aurora kinase A-mediated phosphorylation enhances LDHB activity, promoting tumor progression [261]. In KRAS-driven lung cancer, LDHB tetramers noncanonically block ferroptosis, whereas LDHB suppression activates STAT1 and inhibits SLC7A11-dependent glutathione metabolism [262]. Targeting LDHB preferentially suppresses cancer cell proliferation [263]. In tumor-associated macrophages, LDHB supports a protumor metabolic state by converting lactate to pyruvate [244, 264], and its inhibition disrupts lactic acid metabolism in NSCLC [265].

In the last few years, the roles of LDHC and LDHD in cancer have been explored and found that LDHC promotes breast cancer invasion/migration [266], and correlates with poor progression-free survival [267]. LDHD was generally downregulated in LUAD patients [268], with low expression predicting poor overall survival [269].

The therapeutic potential of LDH inhibition is increasingly evidenced by recent studies. In pancreatic ductal adenocarcinoma (PDAC) models rich in cancer-associated fibroblasts (CAFs), the LDHA inhibitor F×11 has been shown to reduce tumor growth and enhance anti-tumor immunity [270]. Moreover, LDH inhibition demonstrates synergistic effects with IL-2/IL-21 in cancer treatment [64]. Additionally, Berberine, a natural compound functioning as an LDHA inhibitor, suppress the progression of pancreatic adenocarcinoma (Tables 1 and 2) [271].

Mitochondrial oxidative phosphorylation (OXPHOS) enzymes in tumors

OXPHOS in the mitochondrial inner membrane generates the majority of cellular ATP and represents a therapeutic target for various diseases, including cancer [272]. This process is primarily regulated by key enzymes such as NADH dehydrogenase and ATP synthase. NADH dehydrogenase facilitates the transfer of high-energy electrons into the mitochondrial electron transport chain [273]. Polymorphisms in the enzyme have been linked to increased susceptibility to GC and breast cancer in specific populations, such as in Poland [274]. Moreover, elevated expression and activity of NADH dehydrogenase have been shown to promote the tumor growth of neurofibromin-deficient cells [275]. ATP synthase catalyzes the formation of ATP from ADP and inorganic phosphate (Pi), utilizing the proton gradient across the inner mitochondrial membrane. In CRC, the open reading frame within long noncoding RNA LINC00467 encodes a micropeptide named ATP synthase-associated peptide (ASAP). ASAP binds to the α and γ subunits (ATP5A and ATP5C) of ATP synthase, enhancing ATP synthase activity and boosting the mitochondrial oxygen consumption rate, thereby driving tumor cell proliferation [276]. Although the Warburg effect has historically emphasized glycolysis in cancer metabolism, the significance of upregulated OXPHOS in many tumors challenges this longstanding view [277]. Consequently, the role of OXPHOS must be considered in therapeutic strategies targeting glycolytic enzymes, as mitochondrial energy production remains a critical factor in tumor biology and treatment resistance.

Future perspectives

Unresolved issues regarding glycolytic enzymes

Multiple unresolved issues persist in our understanding of glycolytic enzymes in cancer. The roles of HK2 in tumor development appears far more complex than currently recognized. Several critical questions remain unresolved. Take compartmentalization dynamics, context-specific regulation and functional dominance as examples, is HK2 simultaneously expressed across multiple cellular compartments in tumor cells, or is it dynamically recruited to specific localization? In addition, how are HK2’s functions precisely regulated within distinct subcellular compartments? Morevoer, among cytoplastic, mitochondrial and nuclear HK2 pools, which primarily drives tumor growth and metastasis? The recent discovery that palmitoylated HK1 is secreted by hepatic stellate cells under TGF-β stimulation and subsequently hijacked by HCC cells to promote HCC progression [278]. This raises parallel questions for HK2. In the presence of stresses such as chemical drugs, could dying cells secrete HK2 for uptake by viable tumor cells? If secreted HK2 exists, what oncogenic functions might it execute? Furthermore, what are the dichotomous roles of HK2 in tumor cells and tumor-infiltrated immune cells such as CD8⁺ T lymphocytes within the tumor microenviroment? Somatic mutations in PFKP (e.g., R48C, N426S, D564N) are known to alter glycolytic flux through distinct enzymatic effects, yet their functional consequences and mechanistic roles in tumorigenesis remain poorly understood. Hypermethylation of the PFKP promoter region occurs in human epidermal growth factor receptor 2 (HER2) positive breast cancer cells and prostate cancer [279, 280], but its clinical significance requires further validation. Although all of three PFK1 isoforms promote tumor progression, current inhibitors lack isoform selectivity. Given that many normal tissues depend on glycolysis for energy, non-selective inhibition risks significant on-target toxicity. Additionally, it’s proposed that PFKFB3 activity seems localization-dependent. Do different subcellular localizations of PFKFB3 confer distinct pro-tumorigenic functions?

Unlike rate-limiting enzymes (e.g., HK2, PFK1), non-rate-limiting enzymes (e.g., PGAM1, ENO1, LDHA) often exhibit functional redundancy, metabolic flexibility, and tissue-specific isoforms, allowing tumors to bypass inhibition via compensatory pathways. Their limited flux control means inhibiting a single enzyme rarely cripples glycolysis; instead, metabolic rewiring may activate alternative fuels (glutamine, fatty acids) or upregulate parallel glycolytic isoforms. Further complexity arises from their moonlighting functions, creating potential off-target effects upon inhibition. The hypoxic or acidic tumor microenvironment may reverse inhibitor efficacy or amplify resistance. In addition, how can we identify tumors truly “addicted” to specific non-rate-limiting enzymes? Most current glycolytic enzyme inhibitors exhibit low potency and potential systemic toxicity. Can we exploit metabolic heterogeneity without triggering adaptive resistance? Unlocking precision antiglycolytic therapy will require deciphering context-dependent enzyme vulnerabilities and developing isoform-specific agents. Resolving these issues will greatly advance our understanding of glycolysis in cancer and facilitate its clinical translation as a therapeutic target.

The application and challenges of glycolytic enzyme inhibitors

Several inhibitors targeting key glycolytic enzymes have entered clinical evaluation (Table 1). HK2 inhibitors such as 2-DG, 3-BP and arsenic trioxide have been tested in clinical trials. These compounds act through distinct mechanisms. 2-DG, a well-known competitive HK2 inhibitor, targets the glucose-binding site, while 3-BP disrupts the HK2–VDAC1 interaction. ATO induces apoptosis of cancer cells. To date, HK2 represents the most extensively targeted glycolytic enzyme in clinical development, which may be attributed to its organ-specific expression in normal tissues, potentially reducing systemic toxicity risks. In parallel, PFKFB3 inhibitors, such as PFK158 have advanced to Phase I clinical trials. PFK-158 induces apoptosis and holds potential as an anticancer agent. In contrast, PKM2 inhibitors including shikonin, curcumin and proanthocyanidin B2 demonstrate promising antitumor activity but have not yet entered clinical trials.

Among the non-rate-limiting glycolytic enzymes, EGCG targeting PGAM1 and Gossypol targeting LDHA have progressed to clinical trial stage (Tables 1 and 2). Nevertheless, research on targeting non-rate-limiting enzymes remains relatively scarce and predominantly preclinical, underscoring the considerable effort still required for clinical translation, despite their emerging therapeutic relevance.

A major challenge in targeting glycolysis lies in the ubiquitous nature of this pathway across mammalian tissues, raising concerns over inhibitor-related cytotoxicity. For instance, 2-DG may induce metabolic disorders under prolonged or high-dose administration [281]. 3-BP [282] and ATO [283] exhibit dose-dependent hepatotoxicity, and shikonin has been associated with embryonic developmental toxicity and vasodilatory effects [284]. For other compounds such as EGCG, proanthocyanidin B2, and gossypol, comprehensive toxicity profiles are still lacking. Therefore, addressing these safety issues is central to future translational efforts.

Beyond toxicity, several interrelated challenges impede the development of glycolytic enzyme-targeted therapies. A key issue is functional redundancy within the glycolytic network. Non-rate-limiting enzymes often display overlapping roles or are expressed as multiple isoforms. Inhibiting a single non-rate-limiting enzyme may be insufficient to cripple glycolysis, as cancer cells can readily bypass the blockade by upregulating alternative isoforms or activating compensatory metabolic pathways. Furthermore, the ubiquitous nature of glycolysis poses a significant risk for on-target, off-tumor toxicity. Since these enzymes are essential for energy production in normal, proliferating cells; systemic inhibition could lead to severe side effects, which potentially limits the tolerable therapeutic window.

Another layer of complexity arises from the context-dependent, “moonlighting” functions of many of these enzymes. For instance, their roles in regulating apoptosis, immune evasion, or gene expression are often independent of their catalytic activity. This duality means that simply inhibiting the enzyme’s kinase function may not abrogate its full oncogenic potential, and conversely, completely suppressing the protein could have unintended consequences due to the loss of these non-canonical functions. Finally, metabolic crosstalk within the tumor microenvironment further complicates therapeutic targeting. The metabolites such as lactate can shape an immunosuppressive niche, implying that enzyme inhibition may exert unintended effects on stromal and immune cells. Therefore, effectively targeting these enzymes requires a nuanced understanding of how inhibition will impact not only the cancer cell but also the surrounding stromal and immune cells. Overcoming these barriers will require the development of highly specific inhibitors, ideally with isoform selectivity, and combination strategies that account for metabolic plasticity and immune context.

Discussion

Metabolic disorders are a hallmark of numerous malignant diseases, with the conceptualization of cancer as a metabolic disorder tracing back to early observations of its distinctive metabolic features [285]. Consequently, tumor metabolism has become a major focus of research, leading to the classification of various metabolic kinases as oncogenes [286, 287]. This review synthesizes recent advances concerning both rate-limiting (HK, PFK, PK) and non-rate-limiting glycolytic enzymes. Several isoenzymes including HK2, PFKFB3, PKM2, GAPDH, ENO1 and LDH demonstrate tumor-specific expression patterns. Rate-limiting enzymes have received substantial attention due to their pivotal role in controlling glycolytic control. HK2 upregulation not only accelerates glycolysis but also supports tumor initiation and maintenance [12], and its targeting has demonstrated efficacy in ovarian cancer [36], HCC [40], RCC [40] and GM [7]. Similarly, PFKFB3 modulates glycolysis while regulating angiogenesis, cell death, and stemness [288], representing it as a therapeutic target in HCC [113], CML [107] and GC [50]. Aberrant PKM2 expression is a characteristic feature of many tumors [289], and represents a promising target in triple-negative breast cancer [150]. Based on more clinical evidence, we mainly focused on HK2, PFKFB3, PKM2 and MCT-4, and have included relevant mechanistic diagrams to elucidate their roles in tumor biology. In contrast, other glycolytic enzymes have received less emphasis due to the current scarcity of related research, though we have summarized existing findings to support their potential clinical relevance. Additionally, while proteins such as GLUT1 and HK1 are critically involved in glycolysis and tumor progression, their ubiquitous expression in essential normal tissues narrows the therapeutic window and increases the risk of on-target toxicity. Therefore, they are not a focus of discussion in this review.

Non-rate-limiting enzymes also contribute significantly to oncogenesis. Abnormal LDHA upregulation and LDHB downregulation commonly promote aerobic glycolysis and lactate production [290], positioning LDHA as a potential target in PDAC [270] and PAAD [271]. Crucially, many glycolytic enzymes exert tumorigenic effects through non-metabolic functions, which are frequently regulated by subcellular localization or post-translational modifications. Therefore, a comprehensive understanding of the alteration mechanisms—spanning transcription, translation, post-translational modifications, translocation and unique transduction cascade—is essential for designing effective cancer therapies.

This review has highlighted several valuable molecular targets; however, their clinical application faces considerable challenges. Although inhibitors such as 2-DG, PFK158, and lonidamine have demonstrated significant antitumor efficacy in preclinical models, and some (e.g., PFK158) have advanced to Phase I clinical trials, targeted toxicity remains a major concern (Table 2). Given the critical functions of glycolytic enzymes in normal tissues—including the heart and nervous system—systemic inhibition may lead to adverse effects such as cardiac toxicity and neurotoxicity. Key strategies to widen the therapeutic window include developing isoform-specific inhibitors (such as specifically targeting HK2 rather than HK1), allosteric modulators, and targeted delivery systems (such as nanoparticles, antibody-drug conjugates). Additionally, repurposing existing drugs, such as aspirin—which overcomes sorafenib resistance by inhibiting PFKFB3—also holds notable potential.

While this review has centered on glycolytic enzymes, it is important to note that OXPHOS enzymes, including complexes I, II, and III, also exhibit non-classical functions in apoptosis and signal transduction. Future research should adopt an integrated view of tumor metabolism, considering glycolysis and OXPHOS as a functional continuum. The exploration of moonlighting functions of glycolytic enzymes represents a rapidly evolving frontier in cancer metabolism, with profound biological significance and translational implications.

Metabolic disorders, particularly the Warburg effect, are increasingly implicated in a range of diseases [291]. By integrating the mechanistic roles of glycolytic enzymes and their therapeutic targeting, this review advances the understanding of tumor metabolism and provides a foundation for developing novel anticancer strategies.

Abbreviations

GLUT

Glucose transporters

HK

Hexokinase

GPI

Glucose-6-phosphate isomerase

PFK

Phosphofructokinase

TPI

Triosephosphate isomerase

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

PGK

Phosphoglycerate kinase

PGAM

Phosphoglycerate mutase

PK

Pyruvate kinase

LDH

Lactate dehydrogenase

MCT4

Monocarboxylate transporter 4

HCC

Hepatocellular carcinoma

RCC

Renal cell carcinoma

HKDC1

Hexokinase domain protein

G6P

Glucose-6-phosphate

USP

Ubiquitin-specific protease

NK

Natural killer

AML

Acute myeloid leukemia

GM

Glioma

2-DG

2-Deoxyglucose

CQ

Chloroquine

RCC

Renal cell carcinoma

3-BrPA

3-bromopyruvate

F-2,6-BP

Fructose 2,6-bisphosphate

EGFR

Epidermal growth factor receptor

HCC

Hepatocellular carcinoma

CRC

Colorectal cancer

SCLC

Small-cell lung cancer

CTZ

Clotrimazole

GC

Gastric cancer

CSC

Cancer stem cells

PADI

Protein arginine deiminase

DPYSL2

Dihydropyrimidinase like 2

LUAD

Lung adenocarcinoma

LSCC

Laryngeal squamous cell carcinoma

TNBC

Triple-negative breast cancer

CDCA5

Cell division cycle associated 5

KA

Koningic acid

TAK

TGF-β-activated kinase

PDAC

Pancreatic ductal adenocarcinoma

CAF

Cancer-associated fibroblasts

OXPHOS

Mitochondrial oxidative phosphorylation

HER2

Human epidermal growth factor receptor 2

HIF-1α

Hypoxia-inducible factor-1α

mTOR

Mammalian rapamycin targets

VEGF

Vascular endothelial growth factor

CDKs

Cyclin-dependent kinase

CML

Chronic myeloid leukemia

GC

Gastric cancer

ALI

Acute lung injury

ECs

Endothelial cells

SCLC

Small-cell lung cancer

DPYSL

Dihydropyrimidinase like

PAAD

Pancreatic adenocarcinoma

Author contributions

X.C. and J.Y selected the topic. X.C. wrote/original draft preparation. J.Y. and Y.M guided the paper. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (82502218); Zhejiang Provincial Natural Science Foundation of China (LQ24H160040); the Zhejiang Medical and Health Science and Technology Project (2024KY501); Jinhua Municipal Central Hospital Young and Middle-aged Science and Technology Project (JY2022-5-03).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

Not applicable.

Competing interests

The authors declare no competing interests.