In the 1920s, Warburg observed that cancer cells had a distinct metabolism that was highly dependent on glycolysis instead of mitochondrial oxidative phosphorylation, regardless of oxygen availability (1). This process was termed the Warburg effect, or aerobic glycolysis (2). Hanahan and Weinberg (3) also revealed that reprogramming energy metabolism was one of the most common characteristics of cancer cells. Compared with oxidative phosphorylation, glycolysis is a less efficient pathway for producing adenosine triphosphate (ATP) (4). To compensate for the loss of ATP due to preferential glycolysis, cancer cells upregulate genes that encode glucose transporters and glycolytic enzymes, which leads to glucose uptake and an altered metabolism (5). Increased glucose uptake is used in the clinic for the detection of tumors using fluorodeoxyglucose positron emission tomography (4). The metabolic reprogramming of cancer cells provides ATP and allows cells to survive under hypoxic conditions (6). In addition, metabolic reprogramming provides cells with biosynthetic building blocks, including intermediates and substrates for the synthesis of nucleotides, proteins and membrane components, which are required in proliferating cells (7).

Pyruvate kinase (PK) is a rate-limiting glycolytic enzyme that catalyzes the irreversible transphosphorylation between phosphoenolpyruvate (PEP) and adenosine diphosphate, which produces pyruvate and ATP (8). This reaction is a committed step that leads to glycolysis or oxidative phosphorylation of pyruvate (Fig. 1). In mammals, the PK family consists of four isoforms: liver-type PK (PKL); red blood cell PK (PKR); and PK muscle isozyme M1 and M2 (PKM1 and PKM2, respectively). These isoforms are encoded by two genes, PK, liver and red blood cell (PKLR) and PK, muscle (PKM) (9,10). The expression of pyruvates is tissue-specific and regulated by various promoters and alternative splicing. PKL and PKR are products of the PKLR gene. PKL is expressed in the liver, kidneys and intestine and has the lowest affinity to PEP of the isoforms, while PKR is expressed in red blood cells, inhibited by ATP and activated by fructose-1,6-bisphosphate (FBP). PKM1 and PKM2 are produced by alternative splicing of the primary RNA transcript of the PKM gene, which contains sequences encoded by exons 9 and 10, respectively. The non-allosteric isoform PKM1 is constitutively active, and expressed in terminally differentiated tissues, including the muscle and brain, which require a large supply of ATP. By contrast, PKM2 is expressed in tissues with anabolic functions, including proliferating cells and cancer cells, and is subject to complex allosteric regulation. In the majority of cancer cells, the expression of PKM2 is increased, which suggests that PKM2 may be an attractive target for cancer therapy (11). The identification of a novel protein that interacts with PKM2 has confirmed the key function of PKM2 in tumor metabolism and cancer growth (12), revealing novel functions of PKM2 beyond the classical concept of glycolysis.

The PKM2 gene is expressed by alternative splicing of PKM pre-messenger (m)RNA (13). In cancer cells, PKM2 is overexpressed, and overexpression is controlled by the oncoprotein c-Myc. c-Myc activates the transcription of heterogeneous nuclear ribonucleoproteins (hnRNPs) I, A1 and A2, which bind and repress exon 9 encoding RNA sequences (13). This results in the inhibition of PKM1 mRNA splicing, which allows the synchronous expression of the PKM2 isoform (13). A previous study demonstrated that PKM was regulated by the reciprocal effects of the mutually exclusive exons 9 and 10, which lead to the repression of exon 9 and the activation of exon 10 in cancer cells (14). In addition, knockdown of the serine/arginine-rich family of pre-mRNA splicing factor, which reduces lactate production, triggers the expression of exon 10 and promotes the proliferation of cells and aerobic glycolysis. This study suggested that the exonic elements are the actual determinants of PKM2 splicing, as opposed to classical intronic regions.

In the last decade, studies have focused on the metabolism and signal transduction of cells, and have revealed that oncogenes may have direct or indirect associations with metabolic reprogramming (15,16). Mammalian target of rapamycin (mTOR) was demonstrated to be a key activator of the Warbug effect, as it induces PKM2 and other glycolytic enzymes under normoxic conditions (17). Sun et al (17) demonstrated that PKM2 was a crucial glycolytic enzyme in the oncogenic mTOR-induced Warbug effect, in which hypoxia inducible factor-1α (HIF-1α) and c-Myc-hnRNP cascades are the transducers of mTOR regulation of PKM2. Additionally, insulin is closely associated with cancer progression, and also upregulates PKM2 expression through phosphatidylinositide 3-kinase/mTOR mediated HIF-1α induction (18). Notably, the reduction in the activity of PKM2 is independent of this pathway; insulin-induced reactive oxygen species (ROS) was revealed to be responsible (18). Under hypoxic conditions, the PKM2 gene interacts directly with HIF-1α, which activates the hypoxia response element that is required for HIF-1α binding (19).

A previous study demonstrated that the transcription of PKM2 was upregulated by the epidermal growth factor receptor (EGFR) under normoxic conditions (20). EGFR activation induces phospholipase C γ1-dependent protein kinase C (PKC)ε monoubiquitination at lysine-321, which is mediated by RING-finger protein that interacts with C kinase 1 (21,22). Monoubiquitinated PKCε interacts with an ubiquitin-binding domain in the zinc finger domain of IκB kinase (IKK)γ, which leads to the recruitment of cytosolic IKK to the plasma membrane. PKCε phosphorylates IKKβ at serine-177, which activates IKKβ. Activated v-rel avian reticuloendotheliosis viral oncogene homolog A (RelA) interacts with HIF-1α, which is required for the PKM promoter to bind to RelA (21,22). EGFR-promoted glycolysis and tumorigenesis requires PKCε- and nuclear factor κ enhancer binding protein (NFκB)-dependent PKM2 upregulation (21,22). These molecular interactions reveal the importance of the association between EGFR and NFκB pathways in the upregulation of PKM2 and tumorigenesis of cells.

Cells have evolved complex regulatory mechanisms to adapt the metabolism to various physiological states. Rapidly growing cells consume nutrients at a high rate and must maintain a balance between the utilization of nutrients for ATP synthesis and anabolic development, including protein, lipid and nucleic acid synthesis. Cancer cells use glucose at higher rates compared to non-cancerous cells, but use a smaller fraction for oxidative phosphorylation, which enables cancer cells to incorporate a greater fraction of glucose metabolites in macromolecule synthesis instead of expending it on carbon dioxide production (2,3). Consequently, metabolic programming of cancer cells is required to be flexible, which allows the cells to adapt to various environmental conditions. PKM2 is responsible for the final step of glycolysis and is key in this process (2,3). The preferential expression and allosteric enzymatic activity of PKM2 provides the cancer cells with a growth advantage in vivo, without the accumulation of ROS.

Allosteric regulation of PKM2 allows switching between a high- and low-activity state. PKM2 exists in the catalytically distinct tetrameric and dimeric states (2,3). Tetrameric PKM2 exhibits high catalytic activity, which is associated with ATP synthesis and catabolic metabolism in cells. Dimeric PKM2 has low catalytic activity and is the less active state of PKM2 that facilitates the production of glycolytic intermediates to enter the glycolysis branch pathways, including glycerol synthesis and the pentose phosphate pathway, which produces nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) to suppress ROS production and is involved in nucleotide synthesis (2,3).

Various factors have been reported that control the alteration between the dimer and tetramer of PKM2 (Fig. 2) (23). FBP is an upstream glycolytic intermediate and is a well-characterized activator of PKM2, which binds allosterically to PKM2 and facilitates the formation of the active tetramer (11). In addition, serine, which is a glycolytic intermediate 3-phosphoglycerate, is a positive regulator of PKM2 (24). Serine binds and activates PKM2, and a decrease in serine leads to a reduction in the PKM2 activity in cells (24). Phosphoribosylaminoimidazolesuccinocarboxamide (SAICAR) is an intermediate of the de novo purine nucleotide synthesis pathway and also stimulates PKM2 activity (25). SAICAR-PKM2 interaction promotes cancer cell survival in glucose-limited conditions (25–27). This allosteric regulation may lead to cancer cells coordinating various metabolic pathways to optimize cell growth in nutrient-limited conditions. PKM2 activity is also regulated by post-translational modifications, including phosphorylation, acetylation and oxidation, which favor the inactive dimeric state of PKM2 (25–27). Phosphorylation of PKM2 at tyrosine-105 induces the release of FBP, which causes PKM2 to alter between the tetrameric and dimeric states. Additionally, acetylation of PKM2 at lysine-305 downregulates PKM2 activity, which promotes glycolytic pooling, NADPH synthesis and tumor growth (25–27). Notably, a high glucose concentration induces a lysine-305 acetylation of PKM2, which diverts glucose to anabolic synthesis. Oxidation of PKM2 at cysteine-358 confers an advantage to cancer cells, as it allows the cells to withstand ROS (28). Intracellular ROS-induced oxidation of cysteine-358 decreases the activity of PKM2, which diverts glucose flux to the anabolic pentose phosphate pathway, thereby producing sufficient reducing potential for the detoxification of ROS.

Missense mutations in the PKM2 gene, including H391Y and K422R, are observed within the inter-subunit contact domain of the PKM2 protein, which may promote cancer metabolism, oxidative endurance, anchorage independence and tumor growth in a dominant-negative manner (29). The two gene mutations have different effects on the activity of PKM2, which does not affect the stability or expression level of the PKM2 protein. The H391Y mutated isoform of PKM2, resulting from a signal amino acid residue replacement, leads to the total loss of allosteric behavior, which affects the dynamic movement of the protein and results in cell rigidity (29).

Cancer-specific metabolism, which is closely associated with tumorigenicity, cancer aggressive progression and inherent or acquired therapeutic resistance, is an attractive target for cancer therapy. The role of PKM2 in glycolysis and the proliferation of cancer cells has been revealed, and has been demonstrated to balance the production of biomolecular building blocks and the generation of pyruvate and ATP. Consequently, there are two therapeutic strategies for targeting PKM2. The first is inhibitors that block the catalytic activity of PKM2, and the second is activators that induce tetramerization of PKM2 to increase glycolysis.

The present review focused on the PKM2 gene and its regulation, and emphasized the non-metabolic function of PKM2 and the development of PKM2 as a therapeutic target. In the last decade, extensive research has clarified the notable involvement of PKM2 in cancer through its canonical and non-canonical functions. This allosteric property of PKM2 balances cell growth and oxidative stress.

However, PKM2 has multiple roles, and the intracellular mechanisms induced by PKM2 are more complicated than previously hypothesized. Protein kinases are important regulators in numerous biological processes. Numerous human proteins have been identified as potential substrates for PKM2, the majority of which are involved in the regulation of cell proliferation. Additional studies concerning the mechanism of PKM2 substrate recognition may reveal a common mechanism between the proteins involved in cell-cycle regulation. It has been demonstrated that the cell microenvironment provides metabolic heterogeneity in cancer tissues, which may be caused by differences in the supply of nutrients and oxygen (55). Cancer cells are located at various distances from blood vessels, in which PKM2 may play a role. Certain studies suggest that overexpression of PKM2 may be a biomarker for specific types of cancer (56–58); however, this is controversial and additional studies are required for confirmation. The regulation of PKM2 requires additional clarification. Even by providing a functional readout of the oxidative and metabolic state of the cell, it is not fully clear how PKM2 alters the response of the cell to growth factor stimulation (59); however, the association between PKM2 and growth factor responses and oxidative stress responses indicates that this regulation is integrated. It is also contradictory that certain studies report that knockdown of PKM2 results in a modest inhibitory effect on in vitro proliferation of cancer cells and xenograft tumors without signs of inhibition, which indicates that PKM2 is not a requirement for tumor growth (45,46).

In conclusion, PKM2 is essential in the metabolic reprogramming of cancer cells; however, cancer cells reprogram using several metabolic pathways to support cell growth. An improved understanding of metabolism transformation in early cancer cells and how this results in the development and progression of cancer may provide an understanding of the metabolic and non-metabolic functions of PKM2. Furthermore, targeting PKM2 as a treatment for cancer may significantly evolve over the next several years.