Abstract

The metabolism of cancer cells is reprogrammed toward aerobic glycolysis (the Warburg effect), which stimulates tumor growth. Phosphofructokinase (PFK) and its isoforms, PFKP, PFKM, and PFKL, are highly conserved central glycolytic controllers and a potential therapeutic intervention. This review discusses the complex functions of PFK in tumor biology, including its roles in regulating proliferation, invasion, metastasis, and therapy resistance. It further discusses the tumor microenvironmental role of PFK, which influences immune evasion, angiogenesis, and stromal interactions, as well as its non-metabolic signaling functions. The therapeutic approaches to PFK, such as synthetic (e.g., PFK15) and natural (e.g., curcumin) compounds, are considered alongside strategies to address specific difficulties. Lastly, the review is based on a combination of expression analysis of PFK isoforms and a closer analysis of synthetic and natural inhibitors, and it suggests a prospective framework for implementing PFK-targeted therapies in clinical practice that incorporates AI-based drug design, nanodelivery, and immune-metabolic modulation.

1 Introduction

Warburg effect Cancer cells use aerobic glycolysis to augment glucose uptake and utilization, leading to increased glycolysis (De Leon-Oliva et al., 2025). The Warburg effect, first described by Otto Warburg in 1956, is a key feature of cancer metabolism where cancer cells prefer aerobic glycolysis over oxidative phosphorylation (OXPHOS), even when oxygen is plentiful (Warburg, 1956). This metabolic shift supports rapid cell growth, drives uncontrolled proliferation, and creates an acidic, lactate-rich tumor microenvironment (TME) that helps cancer cells evade the immune system, enhances invasion and metastasis, and leads to resistance to standard therapies (Barba et al., 2024; De Leon-Oliva et al., 2025).

Here, cancer cells in cell culture metabolize high glucose concentrations and secrete lactate under high oxygen levels, unlike normal cells, which maintain the balance between glycolysis and OXPHOS (Hammond et al., 2024). The Warburg effect enhances tumor growth and inhibits the immune response by elevating lactate and lowering pH in the TME, thereby impairing T-cells’ function (Barba et al., 2024). The presence of elevated lactate levels also contributes to metastasis by activating pathways, including nuclear factor kappa B (NF-κB) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) (Gupta P. et al., 2014; Barba et al., 2024). Also, acidosis may induce signals leading to cancer cell invasion, especially via the NF-κB pathway (Gupta S. C. et al., 2014). Several studies have reported that the Warburg effect can control the growth of various cancer types, and that disrupting glucose uptake or aerobic glycolysis can slow the tumor’s development (Li et al., 2025).

PFK catalyzes the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate using Mg-adenosine triphosphate (ATP) as a phosphoryl donor in prokaryotic and eukaryotic cells (Kumar et al., 2026). PFK-1, the key enzyme controlling glycolysis, has three tissue-specific isoforms in mammals: platelet type (PFKP), muscle type (PFKM), and liver type (PFKL). Each isoform shows different expression patterns and plays diverse roles in cancers (Lynch et al., 2024; Yuan et al., 2025). The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) family includes four isoenzymes (PFKFB1–4), with PFKFB3 and PFKFB4 being the most overexpressed in human cancers (Kotowski et al., 2021; Zhang et al., 2025). These bifunctional enzymes regulate the intracellular levels of fructose-2,6-bisphosphate (F2,6BP), which is a highly effective allosteric activator of PFK-1. PFKFB3 has the highest kinase activity, promoting glycolysis, whereas PFKFB4 has stronger phosphatase activity, guiding glucose into the pentose phosphate pathway to maintain redox balance (Zhang et al., 2025).

The PFK-1 and PFKFB families are linked through regulated glycolysis to associate with oncogenic signals, stress pathways, and cancer traits. PFKFB3 and PFKP are upregulated by c-MYC, which links proliferation to glycolysis (Liu et al., 2024; Zhang et al., 2025). ROCK1 stabilizes c-MYC in pancreatic cancer to augment PFKFB3 (Pang et al., 2024). PFKFB3, PFKFB4, and PFKP are regulated by hypoxia-inducible factor (HIF-1α) under hypoxic conditions, altering metabolism and leading to tumor resistance (Yi et al., 2019; Kotowski et al., 2021; Zhang L. et al., 2021; Dai T. et al., 2022). The PI3K/AKT/mammalian target of rapamycin (mTOR) pathway promotes glycolysis: AKT phosphorylates PFKFB3 at Ser461 to increase its activity; mTORC1 promotes PFKFB2 to confer cancer stemness (Yi et al., 2019; Fontana et al., 2024; Li et al., 2024). P53 represses PFKFB4, yet p53-deficient cells rely on it to maintain a redox state that becomes vulnerable (Ros et al., 2017). PFKFB3 is phosphorylated by adenosine monophosphate-activated protein kinase (AMPK) under metabolic stress to boost glycolysis and help cells survive mitotic arrest (Doménech et al., 2015). PFKFB3 is approved by the epidermal growth factor receptor (EGFR) pathway and facilitates redox equilibrium, DNA damage repair, and resistance to EGFR inhibitors in non-small cell lung cancer (NSCLC) (Lypova et al., 2024). MiRNAs control the isoforms of PFK: miR-488 silences PFKFB3 and chemoresistance; miR-195-5p silences PFKFB4 (Deng et al., 2021; Sun and Jin, 2022). PFKFB3 is stabilized by ubiquitin-specific peptidase 27 (USP27) in hepatocellular carcinoma (HCC) (Xie et al., 2025); PFKP is degraded by HMG-CoA reductase degradation 1 (HRD1), and its loss enhances breast cancer (BC) aggression (Fan et al., 2021); PFKM is upregulated by Zinc finger E-box binding homeobox-1 (Zhou et al., 2021), which facilitates the Warburg effect and metastasis; PFKM mRNA is altered by N-acetyltransferase-10 and alters glycolysis (Mei et al., 2024). These networks make PFK isoforms critical regulators of metabolism, which present therapeutic targets.

Metabolic targeting in cancer therapy research is also ongoing, aiming to identify small molecules that can suppress key metabolic processes related to cancer growth, especially those in the glycolytic pathway (Pal et al., 2025). This review discusses the functions of the PFK isoforms in cancer development and critically evaluates synthetic and natural PFK inhibitors as therapeutic agents. In addition to listing specific inhibitors, we emphasize that the future of the field lies in capitalizing on the isoform specificity and allosteric regulation of PFK biology, as well as non-metabolic moonlighting functions, to rationally combine and design these compounds into effective anticancer decoys. We also discuss the clinical usefulness of PFK isoforms, current research gaps, and our vision for developing PFK-based precision therapies.

2 Purification and characterization of phosphofructokinase

The key roles of PFK in regulating cancer metabolism have prompted numerous studies on its isolation and biochemical research in different species and tissues. Gradually, purification methods have evolved from simpler salt-based fractionation to more complicated affinity-based purification methods. In the early stages, ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration were used (Kuo et al., 1986; Reibstein et al., 1986). The discovery of affinity chromatography was a breakthrough, more so the utilization of immobilized dyes such as Cibacron Blue F3G-A, which binds to the nucleotide-binding site of the enzyme to purify it with high specificity on sources like yeast, rabbit muscle, and Drosophila melanogaster (Munneke and Collier, 1985; Kuo et al., 1986; Reibstein et al., 1986). Additionally, PFK was isolated and purified from rats’ white and brown adipose tissues using blue dextran-Sepharose, achieving a 1000-fold purification. The enzymes purified from both tissues showed hyperbolic kinetics with fructose 6-phosphate. They were inhibited by ATP and citrate and activated by adenosine monophosphate (AMP), phosphate, and F2,6BP (Sale and Denton, 1985). Human erythrocyte phosphofructokinase was purified 15,000-fold by ammonium sulfate precipitation, heat treatment, and Sepharose 6-B column chromatography, yielding a preparation with a specific activity of 60 µmol of fructose 1,6-bisphosphate formed per minute per milligram of protein at 25 °C (Staal et al., 1972).

The PFK structural analysis has been important in understanding the role and regulation of this protein. The enzyme is usually found as a tetramer (Kuo et al., 1986; Reibstein et al., 1986). Crystallization experiments using human muscle PFK and other microorganisms have produced detailed atomic structures of active and allosteric regulatory sites (Reibstein et al., 1986). Several tissue-specific isozymes, such as muscle (M), liver (L), and platelet (P), have been described, each with specific kinetic and regulatory properties which have been elucidated by cDNA cloning and recombinant methodology (Vora, 1983).

The kinetic and regulatory investigations emphasize that PFK is an allosteric enzyme that is regulated by various cellular metabolites. AMP, adenosine diphosphate (ADP), inorganic phosphate, and F2,6BP are activators that are critical to cause its active R-state, and F2,6BP is the strongest stabilizer (Reibstein et al., 1986; Thomas and Uyeda, 1986; Lynch et al., 2024). In another study, the human muscle PFK was expressed in a yeast strain lacking PFK genes, purified, buffer-exchanged into a specific solution, and concentrated for crystallization. Crystals were grown in a reservoir containing LiNO3, NaF, and sodium acetate at 292 K via vapor diffusion methods, with optimization techniques used to improve quality. Despite success in crystallization, diffraction was limited to 6.0 Å, and crystals showed high solvent content and instability during cryocooling (Kloos et al., 2014).

There are various challenges in purifying PFK because it is highly unstable, prone to aggregation, and likely to be degraded by proteolytic enzymes during extraction, requiring protease inhibitors, substrate analogs, and reducing agents to maintain its functionality (Reibstein et al., 1986). Also, the unique peculiarities of pyrophosphate-dependent PFK isoforms have been exploited to develop sensitive enzyme assays for the detection of F2,6BP (Reibstein et al., 1986; Compton and Patrick, 2025).

Overall, the research and purification of PFK are well established but continue to evolve. The development of affinity purification and structural biology has increased our ability to isolate and study this critical enzyme. Despite ongoing problems with enzyme stability and isoform diversity, modern protocols permit exhaustive biochemical investigation of PFK. This makes it an important area of interest in metabolic research and possible anticancer therapy.

3 The phosphofructokinase endogenous regulators

A system of natural activators and inhibitors tightly regulates PFK activity, thereby maintaining metabolic balance (Table 1). Endogenous activators increase glycolytic flux to meet the cell’s energy demands. AMP and ADP send low-energy signals that stimulate PFK, thereby facilitating ATP generation (Lynch et al., 2024; Wang et al., 2024). This activation is also linked with important cellular signaling pathways: the PI3K/AKT pathway responds to insulin by increasing PFKFB3 levels (Riera et al., 2002; He J. et al., 2025), PFKFB3 is activated by estrogen in cancer cells (Sengupta et al., 2018), and PFK activation is the result of epidermal growth factor (EGF) stimulation using the RAS/RAF/MEK/ERK pathway (Dai S. et al., 2022). Additionally, HIF-1α increases PFKFB3 transcriptional levels (Montemurro et al., 2019).

CategoriesActivators/InhibitorsKey findingsReferencesEndogenous activatorsAMP and ADPSignal low cellular energy states (high AMP/ADP: ATP ratio) to activate PFK and promote ATP synthesisLynch et al. (2024),Wang et al. (2024)Hormonal/Growth factor pathwaysInsulin: Activates via PI3K/AKT, upregulating FKFB3
Estrogen: Stimulates PFKFB3 in cancers (e.g., breast cancer)
EGF: Activates via the RAS/RAF/MEK/ERK pathwayRiera et al. (2002),Sengupta et al. (2018),Dai et al. (2022a),He et al. (2025a)Signaling molecules and transcription factorsHIF-1α: Master hypoxia regulator; transcriptionally increases PFKFB3 expressionMontemurro et al. (2019)Endogenous inhibitorsATPHigh concentrations provide potent feedback inhibition, signaling ample energy availabilityZancan et al. (2008)CitrateKey allosteric inhibitor signaling; abundant biosynthetic precursors (e.g., from the TCA cycle); acts synergistically with ATP.Usenik and Legiša (2010)Acyl-CoAModulates PFK activity through both covalent and noncovalent interactionsJenkins et al. (2011)

Endogenous regulators of phosphofructokinase and their effects.

Endogenous inhibitors are reduced during periods of high levels of energy or when biosynthetic precursors are abundant. The primary ones are ATP, which acts as an inhibitor on its own at high concentrations (Zancan et al., 2008), and citrate, an allosteric inhibitor that suggests there are sufficient biosynthetic building blocks available, and that ATP collaborates with it (Usenik and Legiša, 2010). Moreover, acyl-CoA also influences PFK activity, doing so in both covalent and non-covalent ways (Jenkins et al., 2011).

PFK is one of the enzymes that are important in the glycolysis process, which affects cellular metabolism and energy production. The regulation of its activity by molecules such as F2,6BP and by allosteric effectors such as ATP and citrate illustrates the complexity of metabolite regulation. Insulin- and EGF-induced signaling pathways also underscore the importance of PFK in normal body processes and diseases such as cancer. PFK is associated with multiple pathological conditions when it is not regulated, which explains its possible use as a treatment option. Besides, it is influenced by exogenous influences, such as hypoxia and inflammatory cues, highlighting the importance of the enzyme in metabolic diseases and cancer and the necessity to investigate its regulation in greater detail.

4 A conceptual framework for PFK-targeted cancer therapy

To navigate the maze of PFK biology and its therapeutic manipulation, we suggest a conceptual framework, the PFK-Targeting Ecosystem, that sketches the field into three mutually supporting pillars (Figure 1). Pillar 1: Biological Understanding brings together the basic knowledge needed to determine rational therapeutic points of entry. It consists of tissue-specific expression of PFK isoforms (PFKP, PFKM, and PFKL), allosteric regulation of PFK activity by metabolites (F2,6BP), ATP, and citrate, and upstream oncogenic signaling pathways (HIF-1α, c-MYC, and PI3K/AKT) that regulate PFK expression and activity (Lynch et al., 2024; Yuan et al., 2025). In addition, the downstream effects of PFK-dependent activation, e.g., epithelial-mesenchymal transition (EMT), metastasis, and therapy resistance, along with the newer recognized moonlighting roles of PFK isoforms in nuclear signaling and protein kinase activities, are also included in this pillar (Gao et al., 2021; Wang H. et al., 2023; Paul et al., 2025).

The phosphofructokinase (PFK)-targeting ecosystem. Three pillars of the ecosystem are linked to each other: (1) Biological Understanding - including diversity of PFK isoforms, allosteric regulation, upstream oncogenic drivers, downstream oncogenic processes, and non-metabolic moonlighting activities; (2) Therapeutic Arsenal - such as synthetic small-molecule inhibitors, and natural product-derived compounds, as well as ways to combine them; and (3) Clinical Translation - including predictive biomarkers, advanced nano-delivery systems, and rational combination regimens of these pillars is required to produce next-generation PFK-targeted therapies which are isoform selective, less off-target toxic, and resistant to metabolic plasticity.

Pillar 2: Therapeutic Arsenal lists accessible chemical agents for PFK inhibition, classified as synthetic small molecules (e.g., PFK15, clotrimazole, and 3-bromopyruvate) (Coelho et al., 2011; Guo et al., 2016; Deng et al., 2019; Liu et al., 2019) and natural compounds (e.g., curcumin, resveratrol, epigallocatechin-3-gallate (EGCG), and worenine (Li et al., 2016; Li W. et al., 2020; Ghasemi et al., 2019; Ji et al., 2021). The new combination approaches that exploit synthetic lethality or metabolic vulnerabilities, including the synergistic interaction between PFK15 and metformin (Liu et al., 2019) and imatinib and clotrimazole (Motawi et al., 2015). These are also emphasized in this pillar.

Pillar 3: Clinical Translation outlines the key actions required to bring PFK inhibitors to clinical application. These include the development of predictive biomarkers to stratify patients (e.g., PFKP methylation status and 18F-FDG PET), the design of novel nanodelivery systems to enhance bioavailability and tumor targeting, and the development of combination regimens that rationally combine PFK inhibitors with standard-of-care chemotherapy, radiotherapy, or immunotherapy (Xu Y. et al., 2025).

The main assumption of this framework is that the future of PFK-targeted cancer therapy lies not in the individual achievements of each of the three pillars, but in reflecting on the synergy among all three. Strong biological insights (Pillar 1) should inform inhibitor selection and design (Pillar 2), which should be combined with potent translational strategies (Pillar 3) to achieve clinical success. This review follows this ecosystem, and the latter sections align with each pillar and are completed by a discussion of the integrated future directions.

5 Phosphofructokinase functions in cancer

PFK enables the proliferation, invasion, and migration of cancerous cells, such as prostate, liver, breast, and colorectal (CRC) cancers, as the cancer cells absorb glucose at an accelerated rate and generate lactate, which increases their growth, invasion, and spread (Ma et al., 2025; Wu et al., 2025; Vincken et al., 2026; Zhang et al., 2026). PFK is a vital component of tumor survival and proliferation across different microenvironments, as it plays a central part in metabolic reprogramming. PFKFB3 and PFKFB4 stimulate aerobic glycolysis by generating F2,6BP to activate PFK allosterically. This enhances glycolytic flux, glucose uptake, and lactate production, thereby sustaining the Warburg effect, a metabolic hallmark of cancer, thus boosting rapid biosynthesis and tumor progression (Yang et al., 2025; Zhang et al., 2025; Sharma and An, 2026).

PFKFB3, PFKP, and PFKFB4 collectively promote tumor progression by enhancing cell proliferation, EMT, migration, and invasion. Importantly, PFKFB3 overexpression is linked to EMT activation and resistance to immunotherapy in CRC (Lu S. et al., 2024). PFKP facilitates metastasis via the AXL-MET receptor tyrosine kinase pathway in NSCLC and induces hypoxia-driven glycolytic reprogramming in BC (Zhao et al., 2024; Anwar et al., 2025). Meanwhile, PFKFB4 acts as a key molecule connecting glycolytic activation to EMT-mediated metastatic spread in pancreatic cancer (Lu et al., 2026). In addition, PFKFB3 and PFKFB4 contribute to therapy resistance by conferring resistance to chemotherapy, radiotherapy, and targeted therapy through increased DNA repair, inhibition of apoptosis (through ferroptosis suppression), and stress-adaptive metabolism (He Z. et al., 2025; Wang J. et al., 2025; Vincken et al., 2026). PFK and PFKFB3 control important stromal components in the TME. It enhances protumor polarization and macrophage immunosuppression, promotes metabolic coupling in tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) (Nishi et al., 2025; Shmakova et al., 2025).

In addition to their classical role in glycolysis, the PFK isoforms are also involved in non-metabolic (“moonlighting”) activities that directly affect oncogenic signaling, gene expression, and immune responses. Recent research identified PFKP as a nucleocytoplasmic shuttling protein that contains functional nuclear localization signals and nuclear export sequences (Gao et al., 2021; Liu et al., 2026). Cyclin D3/CDK6-mediated dimerization of the protein facilitates its nuclear entry by exposing the nuclear localization signals and enabling interaction with importin-9 (Liu et al., 2026). PFKP is a transcriptional co-regulator in the nucleus, which increases c-MYC-directed expression of C-X-C chemokine receptor type-4, which facilitates homing and infiltration of leukemia cells into other tissues (Gao et al., 2021; Wang H. et al., 2023). Nuclear PFKP was detected only in invasive T-cell malignancies and not in non-malignant lymphoid tissues; it was also associated with worse patient survival, which suggests it as a diagnostic and therapeutic target (Gao et al., 2021). Equally, PFKFB3 also has functions outside of the production of F2,6BP; it is a signaling scaffold that activates PI3K/AKT/mTOR pathway and Wnt/β-catenin pathways, enhancing p-AKT (Ser473), p-GSK3α/β, and nuclear β-catenin, and anaplastic thyroid carcinoma. The effect of the AKT inhibitor MK2206 reverses these effects, indicating that PFKFB3 is upstream of proliferation and migration and independent of, as well as additive to, its glycolytic activity (Deng et al., 2024). PFKFB3 increases programmed death-ligand-1 (PD-L1) expression on peritumoral monocytes via NF-κB activation, thereby promoting immune evasion by inhibiting cytotoxic T cells in the TME, especially in HCC (Chen et al., 2019). PFKFB4 is an intrinsically active kinase with a non-canonical splice variant (PFKFB4-ΔEx6) that directly interacts with AKT at the kinase domain and phosphorylates it, initiating the PI3K/AKT/mTOR signaling pathway without the need to go through glycolysis. This variant is commonly overexpressed in HCC and is associated with poorer prognosis, and increases the sensitivity of tumor cells to mTOR inhibitors, an example of how a metabolic enzyme can develop a kinase function to be used as a therapeutic target (Wan et al., 2025). Altogether, these moonlighting activities outline PFK isoforms as versatile signaling centers that interconnect metabolic state with transcriptional control, signaling, and immune evasion. This implies that future PFK inhibitors must consider both of these non-canonical protein interactions to develop more selective anticancer therapy that can spare normal tissues.

Besides playing an indispensable role in metabolism, certain PFK isoforms also significantly affect resistance to different cancer therapies. For example, trastuzumab resistance in human epidermal growth factor receptor-2 (HER2)-positive BC and cisplatin resistance in gastric cancer have been linked to overexpression of PFKFB3, which inhibits cell death pathways, such as ferroptosis, or promotes DNA repair (He Z. et al., 2025; Vincken et al., 2026). On the same note, PFKFB4 confers resistance to sunitinib in clear-cell renal cell carcinoma and to lenvatinib in HCC, mainly by engaging alternative survival pathways (Feng et al., 2021; Wang J. et al., 2025). This association between PFK activity and resistance to chemotherapy underscores its potential as a target for enhancing tumor response to therapies. Altogether, PFK is a critical metabolic and signaling pathway in cancer (Table 2), and targeting of this protein represents an attractive method to interfere with tumor metabolism and improve therapy.

Roles in cancerKey mechanismsPFK isoformsReferencesDriver of aerobic glycolysis (Warburg effect)

• Produces F2,6BP to activate PFK, the rate-limiting enzyme allosterically.

• Increases glycolytic flux, glucose uptake, and lactate production.

• Fuels rapid biosynthesis and tumor growth

PFKFB3 and PFKFB4Zhang et al. (2025),2026Promoter of tumor proliferation, invasion, and metastasis

• Enhances cell cycle progression.

• Induces Epithelial-Mesenchymal Transition.

• Increases cell migration and invasion

PFKP
PFKFB3, and PFKFB4Peng et al. (2023),Deng et al. (2024),Lu et al. (2024a),2026; Zhao et al. (2024),Anwar et al. (2025),Chen et al. (2025),Lee et al. (2025),Sun et al. (2025)Contributor to therapy resistance

• Confers resistance to chemotherapy (cisplatin, trastuzumab, and 5-fluorouracil), radiotherapy, and targeted therapies.

• Mechanisms: Enhanced DNA repair, inhibition of apoptosis (e.g., via ferroptosis suppression), metabolic adaptation under stress

PFKFB3 and PFKFB4He et al. (2025b),Wang et al. (2025b),Vincken et al. (2026)Regulator of the tumor microenvironment

• In macrophages Promotes pro-tumor polarization and immunosuppression (e.g., via PD-L1).

• In cancer-associated fibroblasts: Drives metabolic coupling with cancer cells

PFK and PFKFB3Nishi et al. (2025),Shmakova et al. (2025)Involvement in non-metabolic (“Moonlighting”) functions

• PFKP: Can localize to the nucleus and regulate gene transcription.• PFKFB3: Influences key signaling pathways (PI3K/AKT and mTOR).

• PFKFB4: PFKFB4-ΔEx6 directly phosphorylates AKT1 to facilitate the progression of HCC.

PFKP, PFKFB3, and PFKFB4Chen et al. (2019),Gao et al. (2021),Deng et al. (2024),Wan et al. (2025),Liu et al. (2026)

Roles of phosphofructokinase in cancer progression.

The pan-cancer TCGA analysis indicates that the glycolytic enzymes PFKP, PFKM, and PFKL exhibit distinct expression profiles. In PFKP, the expression is highly upregulated in tumor tissues of breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), stomach adenocarcinoma (STAD), but is downregulated in kidney chromophobe (KICH), and kidney renal clear cell carcinoma (KIRC) (Supplementary Figure 1). PFKM shows elevated expression in cancers such as BLCA, BRCA, and cervical squamous cell carcinoma (CESC), and downregulation in CHOL and COAD samples (Supplementary Figure 2). PFKL is generally upregulated, with the greatest upregulation in CHOL, COAD, esophageal carcinoma (ESCA), and STAD, but the lowest levels in thymoma (THYM) and UCEC, and intermediate levels in BRCA and PAAD (Supplementary Figure 3). This heterogeneity underscores that these major metabolic enzymes are tissue-specific, acting as oncogenic activators in some cancers and suppressors in others, underscoring the critical importance of cancer origin in assessing their potential as therapeutic targets.

6 Key genetic and signaling interactions of PFK in cancer

The circuitry of upstream genetic regulators tightly regulates the expression and activity of PFK isoforms, which, in turn, has far-reaching effects on the downstream oncogenic pathways. The key PFK isoforms, PFKFB3, PFKFB4, PFKP, and PFKL, are involved in distinct but overlapping networks of molecular interactions that together drive cancer.

6.1 Upstream regulators

PFK isoforms are regulated by important oncogenic post-transcriptional and transcriptional states upstream (Figure 2). Examples include HIF-1α (Liu et al., 2025), the master regulator c-MYC (Liu et al., 2024), and receptor tyrosine kinase signaling, including the EGFR, all of which can induce PFKFB3 transcriptionally in NSCLC (Lypova et al., 2024). PFKFB3 is also post-transcriptionally regulated by miR-488, which directly binds the 3′-untranslated region of the PFKFB3 mRNA and decreases PFKFB3 protein levels, thereby inhibiting glycolysis and chemoresistance in CRC cells (Deng et al., 2021). In addition, PFKFB3 expression is stabilized by deubiquitinases such as USP27 (Xie et al., 2025). Likewise, PFKFB4 is transcriptionally regulated by the HIF-1α and lysine demethylase-3A/specificity protein 1 (SP1) epigenetic axis (Wang and Wang, 2022), and its mRNA by miR-195-5p (Sun and Jin, 2022). PFKP is regulated by the c-MYC as a transcriptional factor and maintained by the desmosomal protein, plakophilin 1 (PKP1) (Liu et al., 2024; Ritoré-Salazar et al., 2025; Wang et al., 2026), and is degraded by the E3 ligase, HRD1 (Fan et al., 2021). The EMT-transcription factor Zinc finger E-box binding homeobox-1 activates PFKM, while N-acetyltransferase-10 mediates ac4C acetylation and m6A modification via involvement of YTHDC1-LDHA/PFKM, regulating glycolysis (Zhou et al., 2021; Mei et al., 2024). This regulation is a multilayered, dynamically coupled activity of PFK in response to stress signals and the tumor’s metabolic demands.

The PFK Regulatory Hub Network. PFK isoforms (PFKFB3, PFKFB4, PFKP, and PFKL) are linked to upstream oncogenic signaling pathways and downstream cancer-associated programs within the PFK regulatory hub network. Hypoxia-inducible factor-1α (HIF-1α), the master transcriptional regulator c-MYC, receptor tyrosine kinase pathways (such as EGFR), and microRNAs (miR-488 and miR-195-5p) are upstream regulators, as are deubiquitinases (ubiquitin-specific peptidase 27 (USP27)) and protein degradation (HMG-CoA reductase degradation 1 (HRD1)). These regulators focus on individual PFK isoforms, each with its own regulatory landscape. PFKFB3 has isoform-specific downstream effects, including PI3K/AKT/mTOR, Wnt/β-catenin, immune evasion via PD-L1, ferroptosis inhibition by SLC7A11/xCT, and endocrine resistance via ERα. PFKFB4 enhances metastasis by mediating hyaluronan phosphorylation of steroid receptor coactivator-3 (SRC-3) and hyaluronan-mediated extracellular matrix remodeling. PFKP induces a positive feedback loop with c-MYC, triggers the AXL-MET axis, and increases ATP-binding cassette sub-family C member-2 (ABCC2) expression, thereby driving chemoresistance. PFKL improves the fatty acid β-oxidation process through tethering lipid droplets to mitochondria.

6.2 Downstream targets

PFK isoforms coordinate numerous downstream signaling pathways and oncogenic cascades, driving malignant progression (Figure 2). They may be thematically split into four functional modules:

6.3 Basic signal transduction cascades

PFKFB3 serves as a signaling scaffold that activates the PI3K/AKT/mTOR pathway, forming a feed-forward loop that enhances glycolysis and promotes glioblastoma survival (Xia et al., 2025). Meanwhile, PFKFB3 stabilizes β-catenin and facilitates its nuclear entry, thus activating the Wnt/β-catenin pathway and promoting the growth of anaplastic thyroid carcinoma (Deng et al., 2024). PFKP has a positive feedback loop with c-MYC, in which it increases the stability of c-MYC through the ERK mechanism, and c-MYC upregulates PFKP, resulting in the continued proliferative signaling in head and neck squamous cell carcinoma (Liu et al., 2024).

6.4 Immune Evasion

PFKFB3 enhances PD-L1 expression, helping tumor cells evade the immune system. PFKFB3 is overexpressed in CRC, which is a predictor of immunotherapy resistance and an immunosuppressive TME (Lu S. et al., 2024). Moreover, ferroptosis may be prevented by PFKFB3, which can dephosphorylate SLC7A11/xCT at serine 26, thereby sustaining cystine uptake and glutathione production, to cause cisplatin resistance in gastric cancer (He Z. et al., 2025). Besides, PFKFB3 stabilizes estrogen receptor-alpha in ER-positive breast tumors, leading to endocrine resistance in conditions of estrogen deprivation (Jia et al., 2024).

6.5 Differentiation, metastasis, and cellular stress adaptation

PFKFB4 possesses protein kinase activity, and its phosphorylation of steroid receptor coactivator-3 at serine 857 increases its transcriptional activity and gene expression, thereby promoting BC metastasis (Dasgupta et al., 2018). Moreover, PFKFB4 enhances hyaluronan synthesis, which facilitates rearrangement of the extracellular matrix and facilitates invasion (Gao et al., 2018). PFKP also interacts with AXL, activating AXL-MET receptor tyrosine kinase signaling by binding to AXL and facilitating its phosphorylation at Y779, which causes invasive phenotypes in NSCLC (Zhao et al., 2024), as illustrated in Figure 3. PFKL moonlights as a kinase, phosphorylating perilipin-2 on lipid droplets under nutrient-deprived conditions, which promotes lipid droplet-mitochondria tethering. This mechanism facilitates fatty acid mobilization and β-oxidation, another energy source to keep tumor cells alive and growing in HCC (Meng et al., 2024).

Mechanistic diagram of PFKP-driven oncogenic signaling and metabolic changes. PFKP is transcriptionally enhanced by c-MYC and stabilized after translation by plakophilin 1 (PKP1), while HMG-CoA reductase degradation 1 (HRD1) tags it for degradation. It promotes aerobic glycolysis (Warburg effect), supporting biosynthesis and ATP production. Additionally, PFKP activates the AXL-MET axis to facilitate invasion, establishes a positive feedback loop with c-MYC via ERK signaling, increases ATP-binding cassette sub-family C member-2 (ABCC2) expression via NF-κB to confer chemoresistance, and translocates to the nucleus to co-activate C-X-C chemokine receptor type-4 (CXCR4) transcription, aiding tumor cell homing. These functions link together, positioning PFKP as a key regulator of tumor development and therapy resistance.

6.6 Therapy resis