Glycometabolic rearrangements--aerobic glycolysis in pancreatic cancer: causes, characteristics and clinical applications

Carbohydrates are the main ingredient in the daily diet, and cancer applies aerobic glycolysis as a carbon supplement [28, 29]. Glucose is transferred through the cell membrane into the cytoplasm by glucose transporters (GLUTs), including GLUT1, GLUT2, GLUT3, GLUT4, and GLUT5 [30]. GLUT1 and GLUT3 are extensively distributed in all cells as primary transporters to transfer glucose into the cytoplasm [31]. However, most GLUT2 exists in hepatocytes and pancreatic β-cells, and GLUT4 mainly exists in adipocytes and cardiomyocytes, while GLUT5 is distributed in the epithelial cells of the small intestine. Interestingly, GLUT1 is commonly overexpressed in PDAC cells, accompanied by cellular invasiveness induced by the upregulation of matrix metalloproteinase-2 (MMP-2) expression via p53 mutation [32, 33]. Several studies have indicated that MMP-2 overexpression is likely modulated by GLUT1 via the PI3K/AKT and JNK/MEKKI pathways. GLUT1 is transactivated by KRAS, cMyc, and HIF-1α but inhibited by p53. Furthermore, GLUT1 is negatively associated with the expression of PTEN in thyroid cancer cells [31, 32]. Similarly, GLUT3 is activated through the IKK/NF-KB axis and inhibited by p53. Due to the correlation between the overexpression of GLUTs and rising uptake of glucose, glucose can be utilized for positron emission tomography (PET) [32, 33]. Glucose transferred into the cytoplasm by a GLUT is catalyzed by hexokinase (HK) and converted into glucose-6-phosphate. Then, phosphohexose isomerase (PGI) converts glucose-6-phosphate into fructose-6-phosphate (F-6-P). F-6-P is catalyzed by phosphofructokinase (PFK) to generate fructose-1,6-biphosphate (F-1,6-BP) [34]. F-1,6-BP is split into 3-phosphoglyceraldehyde (GA3P) and dihydroxy-acetone phosphate (DHAP). Then, the latter phosphotriose is transformed into the former by triose phosphate isomerase (TPI), and 3-phosphoglyceraldehyde is oxidized to 1,3-diphosphoglyceric acid (1,3-DPG) by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [35] and then catalyzed into 3-phosphoglycerate (3-PG) by phosphoglycerate kinase. 3-PG is converted into 2-PG by phosphoglycerate mutase (PGAM) [36], after which it is dehydrated to form phosphoenolpyruvate (PEP), which is catalyzed by enolase [37]. Following this step, pyruvate kinase catalyzes the transformation of PEP into pyruvate and adenosine triphosphate (ATP) [38]. Then, lactate dehydrogenase (LDH) catalyzes pyruvate conversion into L-lactate, which is the last step of glycolysis [30] (Fig. 2). Glycolysis is the primary metabolic rearrangement in PDAC cells under aerobic conditions, according to Otto Warburg [39].

As continuous upregulation of aerobic glycolysis occurs in PDAC cells under hypoxia/anoxia [45], lactic acid generated from the process of glycolysis causes enormous extracellular and intracellular pH changes caused by hydrogen ions [40]. Normal and stromal tissues will be affected by the acidic microenvironment. Apoptosis and autophagy are induced by acidosis in healthy cells, and the structure of the stromal tissue is modified [96, 97]. However, PDAC cells can survive in this microenvironment due to some adaptive mutations, such as mutations in TP53 and KRAS. The acidic anaerobic microenvironment is a selective pressure that allows most malignant tumor cells to survive in competition for finite substrates and living areas with normal cells, as well as to migrate, invade and metastasize more easily [19, 28, 40, 98].

Interestingly, accumulating literature has demonstrated that oxidative phosphorylation (OXPHOS) is upregulated in some glycolytic cancers, including PDAC, which might be driven by the oncogene KRAS and the loss of LKB1 [99]. According to the reverse Warburg effect, PSCs and CAFs undergo glycolysis and transport metabolites into PDAC cells that are used for mitochondrial respiration to generate energy [84]. Meanwhile, pancreatic cancer stem cells rely on mitochondrial OXPHOS, which may be correlated with the suppression of MYC and the MYC/PGC-1α ratio, so mitochondrial agents and genetic therapy can easily target this phenotype [31]. Nonetheless, some studies have revealed that OXPHOS is not consistently suppressed. Instead, it can be reactivated under some conditions, such as activation of the PI3K-AKT-mTOR and LKB1-AMPK-p53 pathways [99]. As accumulating lactate is released into the microenvironment, glycolysis may be affected by the acidic microenvironment. When glycolysis is inhibited, glycolytic PDAC cells transport pyruvate into the mitochondria for OXPHOS, as the acidified microenvironment makes the reprogrammed microenvironment transport glucose and oxygen more efficiently and easily, which may also be associated with PDAC progression [100]. Intriguingly, mitochondrial OXPHOS accounts for more than 70% of the overall ATP production in cervical and breast cancer cell lines under normoxia, but it is reduced to less than 40% under hypoxic conditions [99]. However, the concrete relationship between OXPHOS and PDAC progression is still unclear, and the specific molecular mechanisms are under investigation. Although the mechanisms are unclear, some data verify that OXPHOS inhibitors can serve as promising therapeutic agents in PDAC [101]. Overall, proliferative PDAC acts as a glycolysis-dominant metabolic cancer with a possible alternative OXPHOS pathway being activated when glycolysis is inhibited. These two metabolic pathways render PDAC aggressive, allowing it to adapt to different microenvironmental conditions. Targeting or inhibiting OXPHOS should be thoroughly considered and verified because the plastic reprogramming of PDAC metabolism may switch OXPHOS cells into more aggressive glycolytic cells.

Immune cells in the cancer microenvironment, including T cells, B cells, natural killer cells, dendritic cells, neutrophils, and macrophages, should convert into glycolytic types to adapt to increasing biosynthetic needs for their anabolic functions and rapid growth in activated states [102‐106]. The secretion of lactate and depletion of glucose by cancer cells can inhibit the functions of immune cells, and PDAC cells are able to escape the immune response [107, 108]. Mechanistically, MCTs exist in stromal cells, such as PSCs and immune cells, as mentioned before; to sustain ceaseless glycolysis in PDAC, the lactate and H⁺ released into the extracellular stroma are transported into immune cells, which induces intracellular acidification and inhibits glycolysis, ultimately resulting in functional damage to the immune cells [109]. Moreover, the lactate levels in PDAC are not as high as we hypothesized, possibly because the abundant CAFs and PSCs as well as immune cells utilize lactate via conversion into pyruvate for OXPHOS [110]. However, regulatory T cells with distinctly lower glycolytic characteristics increase resistance to the low-glucose, high-lactate tumor milieu and retain immunosuppressive functions that may be related to peripheral tolerance in the low-glucose, high-lactate tissue environment [111]. Based on the negative association between glycolysis and immune cells, a novel metabolism-tumor-stroma (MeTS) score was proposed to guide therapy selection for different metabolic classifications. There are four different type: MeTS1, OXPHOS tumors with a high T cell proportion, also called “hot”; MeTS2, reverse Warburg tumor cells with OXPHOS tumor cells and glycolytic stromal cells; MeTS3, a mixed classification having both OXPHOS and glycolytic cells; and MeTS4, glycolytic cancers with a low T cell proportion, also called “cold” [112]. However, not all solid tumors are identified as a specific glycolytic type because of tumor heterogeneity. Furthermore, even in a given tumor, not all cancer cells have the same metabolic identity. PDAC cells have significant glycolytic metabolism features with the reverse Warburg effect observed in PSCs and OXPHOS occurring in PDAC cells, so PDAC should be sorted as MeTS2 or MeTS3 depending on individual characteristics, according to the sorting method.

New publications classify PDAC into 4 subtypes: quiescent, glycolytic, cholesterogenic, and mixed according to metabolic reprogramming features. Among the subtypes, the glycolytic subtype with high expression of glycolysis-related genes exhibits glycolysis-predominant metabolism with the worst clinical outcomes, and the cholesterogenic subtype with high expression of sterol biosynthesis- or cholesterogenesis-related genes promotes pyruvate entry into the mitochondria through mitochondrial pyruvate carriers (MPCs) to produce acetyl CoA for cholesterol biosynthesis and has the best clinical outcomes [113]. The quiescent and mixed subtypes with low and high expression of both metabolic pathways, respectively, show intermediate clinical outcomes. Mechanistically, the glycolytic subtype has a tight correlation with the oncogene KRAS and a weak correlation with MYC. Moreover, HIF1A and related genes, such as LDHA and SLC16A3, exhibit upregulated expression in the glycolytic subtype, which may indicate aggressive features, and the expression of the transcriptional regulator sterol regulatory element binding transcription factor 2 (SREBF2) is upregulated in the cholesterogenic subtype [8]. SREBF2 was proven to attenuate the activation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) promoter, which is upregulated in pancreatic intraepithelial neoplasia driven by KRAS^G12D and may be associated with relatively good outcomes [114].

Intriguingly, almost every enzyme in glycolysis plays a dual-function role during the progression of pancreatic cancer. The enzymes usually perform their catalytic activity in the cytoplasm and regulate transcription factors in the nucleus. The two processes are both crucial in the proliferation, invasion, migration, and metastasis of PDAC, and some new tactics for therapy and detection of PDAC may be developed from this information.

Glucose is converted into glucose-6-phosphate (G-6-P) by HK, which is the first step of glycolysis and rate limiting. HK has four isoenzymes, and glucokinase is the fourth type of HK that mainly exists in the liver and pancreatic β-cells [115]. The product, G-6-P, and long-chain fatty-acyl-coA inhibit the activation of HK. HK2 binds to the outer mitochondrial membrane through voltage-dependent anion channels (VDACs), and HK2 binding to the mitochondria increases the glycolytic capacity and promotes the immortalization of PDAC [27]. Interestingly, the upregulation of HK2 expression is mediated by HIF-1α, as mentioned before. Recently, PTEN/p53-deficient prostate cancer cells were found to exhibit increased expression of HK2, in which PTEN loss activated the AKT-mTORC1-4EBP1 axis to increase HK2 mRNA translation and loss of p53 inhibited miR-143 biogenesis to enhance HK2 mRNA stability. Moreover, miR-34a was shown to directly target HK1 and HK2, which suppressed glycolysis and promoted mitochondrial respiration [35]. Currently, KRAS4A, the unique palmitoylation-depalmitoylation cycle of the RAS isoform, is known to colocalize with HK1 on the outer mitochondrial membrane, and HK1, as a downstream effector of KRAS4A, can enhance glycolytic flux and cancer progression. Intriguingly, KRAS4A displays twice the effect of KRAS4B at the same gene expression level. When HK1 is silenced, however, the difference disappears. This indicates that KRAS4A directly binds to HK1 and acts downstream of KRAS4A to mediate transcription, but palmitoylated KRAS4A inhibits binding with HK1 [116]. Based on the mechanisms linking KRAS4A and HK, targeting KRAS4A to reduce HK levels should be thoroughly studied in clinical research. The expression of HK is linked to increasing glycolysis and an unfavorable clinical outcome in PDAC. 2-Deoxy-D-glucose (2-DG) is an analog of glucose that has recently been used as an HK inhibitor because it can be phosphorylated by HKs but not catalyzed further [117]. Another small molecule, 3-bromopyruvate (3-BP), and the novel agent methyl jasmonate (MJ) can suppress glycolysis and cancer growth in cancer patients [118, 119]. It was concluded that the lncRNA hox transcript antisense RNA (HOTAIR) is tightly linked with HK2 in PDAC patients. Furthermore, HOTAIR promotes the expression of HK2, and HOTAIR and HK2 are overexpressed in both the serum and tumor tissues of PDAC patients [120]. Therefore, their detection may indicate PDAC progression, and targeting HOTAIR to reduce HK2 expression may lead to the discovery of novel therapeutic strategies for PDAC.

Phosphofructokinase (PFK), which catalyzes the second committed step of glycolysis that involves the conversion of F-6-P into F-1,6-BP, is activated by fructose-2,6-biphosphate (F-2,6-BP), which is the strongest allosteric activator of all (including but not limited to ADP, AMP, F-1,6-BP, and F-2,6-BP), and is inhibited by high concentrations of ATP and citrate [28, 34, 121]. Interestingly, F-2,6-BP is catalyzed and hydrolyzed by a dual-function kinase that has two separate catalytic centers, phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFBs) [35]. More precisely, the level of PFKFB3 but not that of other PFKFBs, as a target of HIF-1α that displays the highest phosphofructo-2-kinase activity, is significantly elevated in aggressive cancers, such as PDAC, colon cancer, and breast carcinoma [27, 122]. Similarly, PFKFB4 was also found to have a homologous role in PDAC. PFKFB3 and PFKFB4 are both induced by HIF-1α, hypoxia and augmented expression of the GLUT and VEGF genes [122]. The glucagon-mediated activation of cAMP-dependent protein kinase (AMPK) is responsible for phosphorylation and inducing attenuation of the phosphofructo-2-kinase activity and augmentation of fructose-2,6-biphosphatase activity in PFKFB3 [123]. Additionally, PFKFB3 is located in both the cytoplasm and nucleus, suggesting that it correlates with the proliferation and invasion of PDAC via transcriptional regulatory effects in addition to glycolytic catalysis, similar to other enzymes (pyruvate kinase) in glycolysis. Nuclear localization of PFKFB3 is correlated with enhanced expression of vital cycle proteins, such as cyclin-dependent kinase-1, cyclin-dependent-25c, and cyclin D3, and reduced expression of the cell cycle inhibitor p27 [124]. These specific characteristics of PFKFB3 can be inhibited by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (also known as 3-PO) to downregulate the synthesis of F-2,6-BP and reduce glycolytic flux [125]. It has been suggested that inhibiting the HIF-1α/PFKFB3/PFK-1 axis with metformin could suppress glycolysis and impair cancer growth in hepatocellular carcinoma, but whether similar effects can be achieved in other cancers is not yet clear [126]. Recently, miR-135 expression was shown to be elevated in PDAC as a result of glutamine deprivation. This upregulation is dependent on glutamine deficiency and mutant p53 activated by ROS, which directly promotes miR-135 expression. MiR-135 directly targets PFK expression and leads to decreased mRNA and protein levels, thereby suppressing aerobic glycolysis and increasing OXPHOS flux for attenuation of glutamine dependence to promote PDAC survival in a low-glutamine environment [127]. This information indicates that suppressing miR-135 in the context of enhanced PFK expression and increased glycolysis in PDAC still represses tumor growth. However, we hypothesize that the novel strategy of targeting miR-135 in combination with inhibitors of glutaminase (constructing a glutamine-deficient condition) may create a promising treatment for aggressive PDAC. Similarly, PFK-1 is O-GlcNAcylated at serine 529, which inhibits F-2,6-BP binding with PFK-1 to suppress PFK-1 activity under hypoxia. With reduced PFK-1 activity and glycolytic flux, cancer cells redirect glucose from glycolysis to the pentose phosphate pathway, which shuttles glucose into pathways for protein and DNA synthesis. Conversely, blocking PFK-1 glycosylation at serine 529 impairs cancer formation and proliferation in vivo and in vitro [128].

Notably, pyruvate kinase (PK) catalyzes the conversion of phosphoenolpyruvate into pyruvate and is the last rate-limiting step in glycolysis [35]. F-1,6-BP acts as an allosteric activator of pyruvate kinase. The allosteric inhibitors of pyruvate kinase include acetyl-CoA, ATP, and long-chain fatty acids. Posttranslational modification (PTM) of pyruvate kinase can also regulate its activity. Pyruvate kinase consists of four isoforms: PKLR encodes PKL and PKR; PKM encodes PKM-1 and PKM-2 via a relatively opposite swapped splicing mechanism. PKL is present in the kidneys and healthy liver, PKR exists in erythrocytes, PKM-1 is abundant in differentiated somatic cells, such as brain and muscle cells, and PKM-2 is expressed in fetal tissue and proliferating cells. A previous study showed that PKM-2 is the central kinase in cancer cells in a dimeric form and has no catalytic affinity for phosphoenolpyruvate [30]. In this situation, there are increasing levels of glycolytic intermediates that could be utilized as precursors for metabolic biosynthesis (such as synthesis pathways for lipids, amino acids, and nucleotides), which could perfectly meet the changing growth requirements in PDAC. Mechanistically, PKM-2 catalyzes a reaction in glycolysis and regulates gene transcription related to proliferation and invasion in PDAC, and it has been demonstrated that high polypyrimidine tract binding protein expression promotes PKM splicing to confer drug (gemcitabine) resistance to PDAC cells [38]. With the transformation of PKM-2 from a dimer to a tetramer, PKM-2 has a high affinity for phosphoenolpyruvate, producing a low concentration of metabolic precursors. This transition can be activated by thieno [3,2-b] pyrrole [3,2-d] pyridazinone (TEPP-46), which activates PKM-2 and impairs tumor growth and proliferation, revealing obvious antitumor activity [129]. In addition, PKM-2 induces gemcitabine resistance by downregulating p38 mitogen–activated protein kinase activity, and silencing PKM-2 strongly enhances gemcitabine-resistant cell apoptosis [130].

The lactate dehydrogenase (LDH)-mediated catalysis of pyruvate into lactate is significantly increased in PDAC cells. Previously, the expression of LDH-A (also called LDH-M and encoded by LDHA), which catalyzes the conversion of pyruvate into lactate during the last step in glycolysis, was found to be increased in PDAC and other aggressive tumors. However, the levels of LDH-B (also known as LDH-H and encoded by LDHB), which preferentially performs the reversible conversion of lactate and pyruvate, were also shown to be increased in PDAC [121, 131]. Nevertheless, there is still an argument to be made for targeting LDH-B, which suppresses the glycolytic subtype of PDAC cells and inhibits the proliferation, invasion, and migration of PDAC [132]. Moreover, a clinical study noted that LDH-A in the serum was associated with a poor prognosis after surgery, which might be correlated with a relatively low pH facilitating tumor relapse, as mentioned above (acidosis selects cells that can survive in that microenvironment and leads to mutations, and low pH stimulates tumor invasion and migration) [19, 23, 24]. Another study reported that Forkhead box protein M1 (FOXM1) promotes glycolysis, glucose consumption, and lactate production in PDAC by upregulating LDHA gene expression and augmenting LDHA activity. Unsurprisingly, the FOXM1/LDH-A pathway is also responsible for the progression and growth of PDAC [39]. Targeting LDH-A with siRNA and small molecule inhibitors leads to decreased tumor growth. The selective inhibitor 3-dihydroxy-6-methyl-7-(phenylmethyl)-4-propylnaphthalene-1-carboxylic acid (FX-11) impairs the progression of lymphoma and PDAC xenografts in combination with FK866, a synthetic NAD⁺ inhibitor [133]. Recently, FX-11 in combination with TEPP-46 was employed to treat PDAC cells and demonstrated augmented antitumor activity without obvious toxicity. A phase III trial evaluating the combined treatment has been completed, and the combination is a promising tactic for PDAC therapy [129]. It has been shown that acetylation of LDH-A at lysine 5 (K5) occurs. Acetylation inhibits LDH-A activity, reduces protein levels and promotes the degradation of LDH-A through chaperone-regulated autophagy, which is related to low-efficiency glycolysis by blocking the conversion of pyruvate into lactate, impairing the growth and progression of tumors. Intriguingly, LDH-A strongly accumulates in PDAC, which is accompanied by strongly decreased LDH-A K5 acetylation [134]. Resuming or accelerating the acetylation of K5 in LDH-A in PDAC may produce promising therapeutic results. Novel N-hydroxyindole-based (NHI) inhibitors targeting LDH-A impair proliferation, growth, and migration in PDAC. Moreover, when an NHI inhibitor is synergistically cultured with gemcitabine, it shows enhanced anticancer activity against PDAC [135]. Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor, negatively regulates the transcriptional activity of LDH-A, thus impacting glycolysis and tumorigenesis in PDAC. The KLF4/LDH-A axis has close correlations with glycolysis and the progression of PDAC, as evidenced by both clinical data and experimental data [136].

Aldolase is an enzyme in aerobic glycolysis, and aldolase gene expression is significantly elevated in pancreatic cancer. Aldolase includes three isozymes, aldolase A, aldolase B, and aldolase C, encoded by three different genes, ALDOA, ALDOB, and ALDOC, respectively. Aldolase A is present in muscle tissues; aldolase B is extensively expressed in the kidneys, liver, stomach, and intestine; and aldolase C is expressed in the brain. Remarkably, aldolase A is highly relevant to various malignant cancers, including PDAC, and its expression is significantly increased in metastatic PDAC, in which it is used as a crucial indicator for detection. After treatment with transforming growth factor-β, PANC-1 cells exhibit increased ALDOA expression, leading to enhanced glycolysis. Furthermore, silencing aldolase A decreases aerobic glycolysis and ROS generation and inhibits the proliferation and metastasis (as indicated by EMT markers such as E-cadherin, N-cadherin, and vimentin) of PDAC in vivo and in vitro [137]. Similar to other enzymes involved in glycolysis, aldolase A is speculated to play dual-function roles in PDAC: catalysis in the cytoplasm and transcriptional regulation in the nucleus, which has been clarified in the pathogen Francisella [138]. A study showed that aldolase A could be inhibited by the hypoxic cytotoxin 3-[2-hydroxyethyl (methyl)amino]-2-quinoxalinecarbonitrile 1,4-dioxide (TX-2098). TX-2098 treatment suppressed the expression of HIF-1α, vascular endothelial cell growth factor, GLUT1, and aldolase A, leading to distinct antitumor efficacy in a xenograft PDAC model [139]. Moreover, naphthalene-2,6-diyl bisphosphate (ND1) is an active site substrate mimic that acts as an effective inhibitor of aldolase A, but it can be hydrolyzed easily. To address this shortcoming, a series of analogs of ND1 were extensively researched to identify covalent and noncovalent inhibitors. The most stable noncovalent inhibitor identified is NDB, which has two difluoromethylene insertions that do not impair the binding affinity. However, biochemical assays showed that methylene insertion weakened the effect of ND1 [140]. It is clear that the lncRNA DIO3OS promotes the growth of PDAC tumors with low expression of miR-122 and high expression of aldolase A [141]. Therefore, the DIO3OS/miR-122/aldolase A axis could be exploited as a therapeutic target in PDAC progression.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), another vital enzyme in glycolysis, exhibits increased expression in PDAC at both the mRNA and protein levels. Acetylation of lysine 254 augments the activity of GAPDH in glycolysis and promotes the proliferation of cancer cells [35]. Increased GAPDH mRNA and protein expression is associated with increased glycolytic flux in PDAC. It was demonstrated that GAPDH exerts its effects through DNA repair, autophagy, apoptosis, iron metabolism and transcriptional regulation in addition to catalysis in glycolysis, so this enzyme can be detected in the cytosol, membrane, nucleus, Golgi, and endoplasmic reticulum. Moreover, mutant p53 enhances glycolytic GAPDH activity and induces the formation of the SIRT1-GAPDH complex, which can stabilize cytosolic GAPDH for glycolysis and induce tumor growth and survival in PDAC [10]. The natural product koningic acid (KA) is an established selective inhibitor of GAPDH that exerts bioactivity mainly on tumors, with little effect on normal tissue. It inhibits the activity of GAPDH, leading to low glycolytic flux and a low cytotoxic response in highly glycolytic tumor cells [142]. Another inhibitor of GAPDH is iodoacetate; when PANC-1 cells are cultured with iodoacetate, low glycolytic flux is observed, accompanied by reduced cell survival. However, there is little alteration in the signaling machinery [143].

Phosphoglycerate kinase (PGK), as the first enzyme in glycolysis that produces ATP, includes two forms of PGK, the extensively expressed form PGK-1 and the testis-expressed form PGK-2. It has been demonstrated by immunohistochemistry that PGK-1 is highly expressed in PDAC [144]. Interestingly, PGK-1 has been proven to affect DNA duplication and repair in the mammalian nucleus. The mRNA and protein expression of PGK-1 is relevant to poor outcomes and a poor prognosis [145]. Accordingly, PTMs play vital roles in regulating the function of PGK-1 during tumorigenesis. More specifically, promoter methylation has been negatively associated with the mRNA expression of PGK-1; phosphorylation of the PGK-1 protein was associated with the clinical outcomes of PDAC patients [144]. Interestingly, another study reported that pyruvate dehydrogenase kinase 1 (PDHK-1) is phosphorylated by mitochondria-translocated PGK-1 (acting as a protein kinase), which activates PDHK-1 and augments glycolysis but inhibits OXPHOS [146]. Under glutamine deprivation and hypoxia in cancers, PGK-1 has an enhanced interaction with the acetyl-transferase ARD1, leading to acetylation of PGK-1 at lysine388. Subsequently, PGK-1 (acting as a protein kinase) phosphorylates Beclin1 without affecting the formation of Beclin1/VPS34/ATG14L, which causes an altered conformation and increased activity of VPS34 as well as augmented autophagy for tumor homeostasis [147]. However, HIF-1α upregulates the expression of PGK-1 but not its subcellular distribution under hypoxia. Increased expression of PGK-1 can result in the catalytic phosphorylation of troxacitabine, converting troxacitabine into the triphosphate form and causing increasing cytotoxicity to PANC-1 cells [148]. When PDAC is treated with troxacitabine, creating PGK-1 overexpression conditions to increase the cytotoxicity of troxacitabine may be a potent method for PDAC therapy. In a study, when FOXM1 expression was knocked down, PGK-1 levels were also decreased significantly, similar to the changes in LDHA levels mentioned before; however, the author did not specify the concrete mechanisms related to FOXM1 and PGK-1 [39]. Nuclear Factor of Activated T Cells 5 is highly expressed in PDAC patients and is associated with tumor progression by positively modulating PGK-1 [149]. SMAD4 and PTEN are distinctly silenced in PDAC. Loss of SMAD4 in PDAC is responsible for the high glycolytic capacity and tumor progression by upregulating PGK-1 expression, which has been shown to have dual roles in glycolytic catalysis and transcription factor activity in metastasis. Nuclear PGK-1 induces EMT by repressing E-cadherin expression, thus contributing to the migratory and metastatic potential. Cytoplasmic PGK-1 affects the metabolic type of PDAC by regulating the ratio of glycolysis and mitochondrial oxidative phosphorylation. Moreover, SMAD4-silenced PDAC patients can be classified by the subcellular localization of PGK-1 into nuclear PGK-1 positive or negative and high or low cytoplasmic PGK-1 to differentiate patient prognoses [150]. The new classification and interaction between SMAD4 and PGK-1 suggests some predictions and an instructive strategy for PDAC treatment, but these conclusions need to be thoroughly confirmed before a clinical application is proposed. In addition, PGK-1 is activated through autophosphorylation at tyrosine 324 (Y324), which enhances glycolysis and proliferation. PTEN exerts its protein phosphatase effect and dephosphorylates PGK-1, but the loss of PTEN found in glioblastoma is strongly associated with a poor prognosis [151]. Thus, regulating the PTMs of PGK-1 to block glycolysis and proliferation in tumors may provide novel methods for cancer therapy.

Phosphoglycerate mutase (PGAM) displays relatively high expression and activities in many malignant cancers, including PDAC. Via proteomic techniques, PGAM-1 is statistically indicated to be associated with a relatively poor prognosis in PDAC [152]. After knockdown of PGAM-1 expression, decreased glycolytic flux and reduced cell invasion were observed by detecting 3-PG and 2-PG levels and performing invasion experiments, respectively, but the association with proliferation was independent [36]. A study reported that PGAM-1, which is downstream of the PI3K/Akt/mTOR pathway, promoted EMT by stimulating the Wnt/β-catenin pathway, offering a potential application in PDAC through targeting a related signaling pathway to regulate the activity or expression of PGAM [153]. Moreover, acetylated PGAM-1 displays augmented enzymatic activity, and SIRT-1, an NAD⁺-dependent deacetylase, inhibits enzymatic activity by deacetylating PGAM [154]. Another NAD⁺-dependent deacetylase, SIRT-2, induces deacetylation at lysines 100/106/113/138 of PGAM-2 to inhibit enzymatic activity and repress tumor proliferation [155]. Interestingly, the phosphorylation of PGAM-1 at tyrosine 26 (Y26) was proven to activate enzymatic activity to alter glycolytic flux and induce migration [156]. Therefore, targeting PTMs, such as dephosphorylation and deacetylation, at some sites in PGAM-1 could attenuate PGAM-1 enzymatic activity and tumorigenesis for PDAC treatment. Recently, a newly developed and promising compound, KH3, was found to act as an allosteric suppressor of PGAM-1, showing satisfactory drug effects to downregulate glycolysis and cell proliferation with limited cytotoxicity to PDAC cells [152].

Enolase is the enzyme that generates PEP in glycolysis, and there are five forms of enolase in mammalian tissue composed of three immunological subunits, alpha, beta, and gamma. The alpha subunit exists in many tissues; the beta subunit is present only in skeletal and heart muscles, and the gamma subunit is localized in neurons [157]. Alpha enolase (ENO-1) levels are elevated in PDAC cells [37, 157, 158], which correlates with the migration and invasion of PDAC cells by inducing plasminogen activation into plasmin to degrade the compact ECM in a plasminogen-dependent process, and this process can be strongly blocked by using an adeno-associated virus/anti-ENO-1 antibody construct [159]. In addition, aberrant expression of ENO-1 is positively correlated with Ki67 and negatively correlated with p53 in PDAC, which indicates that ENO-1 exerts its effects on proliferation and metastasis by regulating the Ki67 and p53 pathways. In addition, increased ENO-1 expression is relevant to HIF-1α under hypoxia [37]. ENO-1 acts as the receptor of plasminogen in addition to a glycolytic enzyme, and silencing ENO-1 attenuates adhesion, invasion, and metastasis in PDAC [160]. Similarly, PTMs can also regulate ENO-1 activity, and phosphorylating ENO-1 peptide-MHC complexes at serine 419 can induce T cell signaling and autoantibody production in PDAC [158]. Accordingly, SF2312 produced by Micromonospora actinomycetes was first used as an antibiotic and is considered a specific inhibitor of enolase [161]. ENO-1 DNA vaccination has been established for PDAC, and additional treatments, such as ENO-1 inhibitor application, immune cell activation, or chemotherapy, in combination with ENO-1 vaccination could amplify the therapeutic response in PDAC [162].

The oxidative phenotype exists in PDAC, and cells with the glycolytic phenotype can also choose OXPHOS to sustain metabolic needs when glycolysis is inhibited in PDAC, as we mentioned before. Increased OXPHOS can be characterized by activated enzymes and increasing levels of products of the TCA cycle; moreover, mitochondrial dynamics and the mitochondrial membrane potential can be evaluated to confirm this phenotype [100]. The ROS level is the major factor that affects the tumorigenesis of PDAC, and low levels of ROS, which are produced by respiratory complex IV, activate vital redox signaling pathways; additionally, respiratory complexes I, II, and III produce superoxide, which may cause oxidative stress and mitochondrial dysfunction [163]. Mitochondrial respiration unifies several important bioenergetic pathways; it produces precursors for lipid, amino acid, and nucleotide biosynthesis and contributes to glutamine metabolism (Fig. 2). In addition to glucose, glutamine, a dispensable amino acid, is another nutrient fuel for cancer energy production and biosynthesis. Of note, glutamine deprivation is greatly associated with tumor suppression [164]. Glutamine is transferred into cells through alanine/serine/cysteine-preferring transporter 2 (ASCT2) in PDAC cells [165], and then it is converted into glutamate by glutaminase (correlated with EMT in hepatocellular carcinoma and encoded by GLS1 [166]) and metabolized to α-ketoglutarate as a carbon donor for the TCA cycle via amino acid transaminase or glutamate dehydrogenase, which is also accepted as anaplerosis. In addition, glutamine provides nitrogen for nucleotide synthesis and synthesis of other dispensable amino acids (such as arginine, asparagine, serine, alanine, aspartate, glycine, and cysteine) [167]. De novo lipogenesis is a prominent feature of PDAC cells and is essential for fatty acid biosynthesis. Lipid metabolism provides aggressive PDAC cells with enough lipids for the cell membrane, energy production, and second messenger and signaling molecule generation. Another intermediate in the TCA cycle, citrate, is transformed into acetyl-coenzyme A (CoA) via ATP-citrate lyase (ACLY) and then converted into malonyl-CoA, which is mediated by acetyl-CoA carboxylase (ACC) (encoded by ACACA or ACACB), finally producing fatty acids through fatty acid synthesis (FASN). The expression of ACLY, ACC, and FASN is augmented in some PDAC patients, who show a shortened survival time, chemoresistance, and a poor prognosis. Downregulating the activity and expression of these proteins reduces lipid generation while suppressing tumor proliferation and inhibiting tumorigenesis [168‐170]. In hepatocellular carcinoma, it has been proven that inhibiting the AMPK-induced phosphorylation of ACC causes larger lesions and progression in hepatocellular carcinoma. The opposite results are observed when treatment with the ACC inhibitor ND-654, which imitates the effects of phosphorylating ACC, is applied [171]. Expression of FASN is mediated by transcriptional factor sterol regulatory element-binding protein 1c (SREBP1c), and downregulating PI3K and MAPK pathways can inhibit SREBP1c to decrease FASN transcription and suppress lipogenesis and PDAC progression. Furthermore, after FASN and SREBP1c expression was analyzed, SREBP1c was demonstrated to directly or coordinately regulate enzymes and FASN in lipogenesis [172]. The hexosamine biosynthetic pathway (HBP) is greatly interlinked with other metabolic pathways, such as glucose, lipid, nucleotide, and amino acid metabolism, in PDAC. The end product of the HBP, uridine diphosphate N-acetyl glucosamine (UDP-GlcNAc), is generated from 3 materials, glucose, glutamine, and glucosamine, and acts as a substrate for O-GlcNAcylation. The HBP and glycolysis share the first two steps from glucose to F-6-P generation, and then F-6-P and glutamine are converted into glucosamine-6-phosphate via glutamine fructose-6-phosphate amidotransferase (GFAT); meanwhile, glucosamine is converted into glucosamine-6-phosphate by GlcNAc kinase. Then, glucosamine-6-phosphate is catalyzed to UDP-GlcNAc with the assistance of intermediates including acetyl-CoA and UTP from de novo lipogenesis and nucleotide metabolism, respectively [173]. O-GlcNAcylation is a PTM on some proteins that leads to metastatic potential and aggressive reactions, and transcriptional regulation is widely disrupted (both O-GlcNAcase and O-GlcNAc transferase levels are increased) in PDAC to promote tumor growth [174]. The activated HBP is not only highly interconnected with other metabolic pathways but is also closely related to growth, PTMs, invasion, EMT, and aggressiveness in PDAC [173].

Recent detection approaches for PDAC include biomarker evaluations, imaging assays, and oncogene mutation evaluations, but none of these can act as an independent confirmatory diagnostic measurement. The anatomically inaccessible location of the pancreas leads to an incomplete diagnosis [29]. Some serum biomarkers, such as CA-125/CEA/CA19–9, are correlated with poor surgical outcomes and a poor prognosis [175‐177]. However, conventional tumor markers, such as CA19–9 and CEA, lack sensitivity and specificity. However, traditional imaging examinations such as CT and magnetic resonance imaging (MRI) can only reflect the visual tumor size measured by a computer [178]. These measurements lack the foresight needed to predict the occurrence of PDAC and provide additional information related to malignancy. Thus, analysis of metabolic rearrangements in glycolysis could be considered a relatively precise diagnostic means for the detection of the metabolic phenotypes of pancreatic cancer to indicate malignancy. Specifically, this section summarizes alterations in enzymes and serum metabolites in glycolysis differing between PDAC patients and healthy people to explore if these parameters could be prospective measurements for PDAC (Table 1).

Augmented enzyme expression and activity indicate an increase in glycolysis, which also acts as a vital sign of PDAC [202]. These changes in proteins can be detected by mass spectrometry (MS), which identifies molecules by measuring the mass-to-charge ratio, and the instrument includes an ion source, a mass analyzer, a detector, and a data system [203]. Moreover, GLUTs accumulate in PDAC, so more glucose is conveyed into the cytoplasm through these transporters. Upregulation of GLUT expression is generally associated with a poor prognosis and can be detected by the MS technique [26, 204]. Similarly, this tool has been developed to analyze proteomics, including large-scale protein expression and PTMs in glucose metabolism. Dysfunction of the enzymes in glycolysis is usually induced by PTMs, which are involved in the formation, proliferation, and invasion of PDAC, such as acetylation, phosphorylation, O-methylation, and glycosylation, and these modifications can also be detected by LC-MS/MS. [205] However, not all molecules can be detected by MS, and biomarkers in glycolysis with a low abundance in PDAC, such as HIF-1α, cannot be detected by MS. [26].

Presently, many researchers have deemed metabolism to be a vital characteristic for detecting and treating pancreatic cancer, so the metabolic tumor burden (MTB), especially total lesion glycolysis (TLG), is a new parameter to discriminate pancreatic carcinoma from benign pancreatic diseases. In addition to discriminating PDAC, TLG can also predict clinical outcomes and prognosis because it is related to poor OS and recurrence-free survival (RFS) in PDAC [206]. Interestingly, these indexes, such as the MTB and metabolic tumor volume (MTV), are detectable by 18-fluoro-deoxyglucose positron emission tomography computed tomography (¹⁸FDG-PET/CT) [20], which uses ¹⁸FDG as a tracer because of the elevated uptake of glucose. Notably, ¹³C-labeled metabolites can translate into cancer cells for reprogramming metabolism, such as glycolysis, and the metabolic process can be detected by hyperpolarized magnetic resonance spectroscopy (MRS), which can be used to discover multiple metabolic processes via labeled metabolites of glucose, amino acids, lipids, and nucleotides in PDAC [29]. For example, 1-¹³C-labeled pyruvate was converted into lactate in a preclinical PDAC model and detected by hyperpolarized MRS to discriminate cancer cells from normal cells [207]. The safety of hyperpolarized MRS has been proven in clinical trials for prostate cancer [208]. In addition, a new study reported that the specific preoperative neutrophil-lymphocyte ratio in the serum was associated with OS after resection in PDAC patients [209].

.In summary, conventional imaging examinations such as CT and MRI can be used for surgical resectability and range; biomarkers in body fluids, such as the serum and urine, can be evaluated to monitor for PDAC; the expression of glycolytic enzymes and regulators and the concentrations of glycolytic metabolites in the serum can be used to predict prognosis and malignancy; PET and MRS can be used to evaluate the metabolic properties and metastasis of pancreatic cancer; and TLG and MTV can be used to predict prognosis and clinical outcomes. We believe that an integrated method together with genomic profiling of PDAC may provide a prognostic and personalized diagnosis for PDAC patients.

Traditional treatment strategies include chemotherapy, radiation therapy, and molecular targeted therapy. However, surgery is the only possibly curative treatment [210]. After surgery, neoadjuvant chemoradiation is increasingly used to treat PDAC patients [211]. Furthermore, metabolic rearrangements can not only reflect characteristics and hallmarks for diagnosis and prognosis but also lead to the development of new therapeutic strategies as biomarkers for PDAC. This review summarizes inhibitors of glycolytic enzymes and regulators to explore whether they could be prospective treatments for PDAC (Table 2).

Recently, the fructo-1,6-bisphosphatase (FBP)-mediated rate-limiting step in gluconeogenesis has been reported to inhibit the Warburg effect and KRAS signaling. Furthermore, FBP-2 and FBP-1 were found to attenuate soft tissue sarcomas (STS) and breast cancer, respectively. In STS, the authors re-expressed FBP-2 through overexpression vectors and found that FBP-2 inhibited glycolysis in the cytoplasm and suppressed mitochondrial respiration, biogenesis and the tricarboxylic acid (TCA) cycle in the nucleus by inhibiting the function of the transcription factor c-Myc in the nucleus. In breast cancer, restoration of FBP-1 expression intensely inhibited glycolysis by catalyzing the function and silencing activity of HIF [34]. However, the exact role of FBP-2 in PDAC is still obscure; the precise mechanism remains unknown. In addition, targeted therapy aimed at common gene mutations consistently exhibits a lack of efficacy because the most commonly mutated genes in pancreatic cancer are known to be KRAS and TP53, which can hardly be made into drug targets [3]. Promisingly, AMG-510, the first inhibitor of KRAS in clinical development, caused a decline in KRAS^G12C tumor growth and augmented the efficacy of chemotherapy and targeted agents [213]. Moreover, in clinical data, approximately 8% of PDAC samples had germline and somatic mutations in the DNA damage repair genes BRCA2, PALB2, and ATM [6]. Inspiringly, a recent study noted that olaparib, a poly ADP-ribose polymerase (PARP) inhibitor, prolonged PFS among germline BRCA-mutant PDAC patients [214‐216]. Olaparib has been approved by the FDA for metastatic pancreatic cancer, breast cancer and recurrent ovarian cancer with BRCA1/2 mutation [214]. Although olaparib exhibits a therapeutic effect, the application of olaparib is limited to patients with mutations in the DNA damage repair gene BRCA2, and further studies will be required to identify a detailed approach for treating PDAC.