As opposed to normal, differentiated cells, which under aerobic conditions metabolize glucose mainly via oxidative phosphorylation (OXPHOS), cancer cells largely favor glycolytic pathway and subsequent lactate¹ formation for their energy production, regardless of oxygen availability. Warburg first observed this metabolic peculiarity of cancer cells (1) and postulated not only that cancer cells have damaged respiration and excessive glycolysis but also that the shift of energy metabolism from aerobic to anaerobic is actually the cause of cancer (1). According to Warburg, the tumor is initiated by irreversible damage to respiration and persists because of increased anaerobic metabolism, which compensates energetically for the failure of respiration (1). However, today we know that many cancer cells have healthy mitochondria (2) and rely partly on oxidative metabolism (3), whereas fermentative glycolysis remains the “preferred” pathway by most hypoxic and rapidly growing tumors (4–6).

Following these pioneering studies, the field of cancer metabolism has been in a shadow of cancer genetics, which prevailed for decades, after the discovery of the role of oncogenes and tumor-suppressor genes in cancer. However, in the late 1990s, it was shown that lactate dehydrogenase A (LDHA) is a direct c-Myc-responsive gene (7), followed later on by the discovery that c-Myc and HIF-1 complementary induce all glycolytic enzymes with a concomitant inhibition of the pyruvate oxidation (8), reviving interest in connecting oncogenes and altered metabolism (4). At this time, altered metabolism was seen only as a consequence of oncogenic activation, since serum growth factors known to rapidly activate metabolism in the early 1970s (9) were shown to induce c-Myc. Interestingly, it was shown only later that loss-of-function mutations of the TCA cycle enzymes succinate dehydrogenase (10) and fumarate hydratase (11) were implicated in pathogenesis of several hereditary forms of cancer. These mutations in tumor-suppressor genes encoding for important metabolic enzymes raised the possibility that under certain conditions, altered metabolism could be the cause, not the effect, of cancer transformation (12).

Even if seemingly counterintuitive, given the much lower ATP yield from glycolysis with respect to the OXPHOS, this reprogramming of energy metabolism is thought to support large-scale macromolecule biosynthesis, necessary for rapid proliferation and growth (5, 6, 13) (Figure 1). Metabolic rearrangements are a feature of almost all cancer cells, which enables them to adapt to constantly changing conditions in nutrient microenvironment thereby promoting their aberrant proliferation. Aerobic glycolysis (Warburg effect) is just one component of the metabolic transformation, together with the reverse Warburg effect (14), metabolic symbiosis (15) and addiction to glutamine metabolism (16).

In this mini-review, we report the tumor growth consequences of re-routing fermentative glycolysis by genetic disruption at three key levels studied in our lab: glucose-6-phosphate isomerase (GPI), lactate dehydrogenase (LDHA and B isoforms), and at the level of export of lactic acid [monocarboxylate transporter (MCT) isoforms]. We discuss their responses in terms of metabolic rewiring, growth arrest, or tumor escape and compare it with a broader literature.

Despite the remarkable genetic and phenotypic tumor heterogeneity, a specific set of signaling pathways appear to support the altered metabolic processing of glucose. Indeed, there is a dual set of universal mitogenic pathways: Ras-Raf-ERK and PI3K-AKT activated by growth factors/hormone receptor tyrosine kinases and G protein-coupled receptors. ERKs and AKTs protein kinases synergize in controlling growth and metabolism through activation of the master protein kinase (mTORC1). In cancer, oncogenes and tumors suppressors constitutively activate these mitogenic pathways to modify metabolism, nutrient, and oxygen sensing through c-Myc and HIF-1 (17–19). Regulation of cancer cells’ metabolic rearrangements by oncogenes and tumor suppressors is complex and beyond the scope of this short review, but the fact that numerous pathways converge on glucose and glutamine reflects their central importance for energy metabolism.

The avidity of cancer cells for glucose is reflected by the upregulation of glucose transporters and clinical exploitation of the accumulation of radioactive ¹⁸F-deoxyglucose is identified by positron emission tomography. Once inside the cell, glucose is metabolized by glycolysis, a pathway embedded in a complex metabolic network, directly providing precursors for nonessential amino acids (20) and through branching to the oxidative arm of pentose phosphate pathway (PPP), nucleotides (20) (Figure 1). Furthermore, NADPH is regenerated in the PPP and by the serine, glycine/C1-carbon synthesis glycolytic bypass thus contributing to reductive biosynthesis and redox homeostasis (21). As such, branching of the glycolytic pathway is strictly regulated at several different steps (22).

Recognition that the oncogenic activation leads to increased glycolysis (23), together with clinical evidence that correlated cell metabolism with cancer outcome, prompted many studies toward strategies to inhibit glucose metabolism in cancer (24, 25). In fact, some of the first metabolic anticancer therapies developed remain effective agents in clinic today, such as antifolate drugs and l-asparaginase (25). 2-deoxy-glucose (2-DG) has been recognized as a glycolysis inhibitor since the 1950s (26, 27), primarily by competitively inhibiting GPI (26, 28). However, 2-DG also inhibits glucose transport (29), hexokinase (HK) activity (30, 31) and the multiple points of action and its high toxicity have prevented its use in the clinic (32, 33).

Glucose-6-phosphate isomerase (d-glucose-6-phosphate aldose-ketose-isomerase; EC 5.3.1.9) is a housekeeping cytosolic enzyme that plays a key role in glycolytic and gluconeogenic pathways, catalyzing the interconversion between G6P and fructose-6-phosphate (Figure 1). Its expression is induced by c-Myc (34) and HIF-1 (35, 36) and is increased in many cancers (37). GPI has also been described as a secreted multifunctional complex protein that could act as a cytokine under the name autocrine motility factor (38). However, this notion requires further confirmation.

In our lab a complete genetic ablation of GPI expression was accomplished by using CRISPR/Cas9 in two aggressive cancer cell lines, human colon adenocarcinoma (LS174T) and mouse melanoma (B16-F10) (39). Both GPI-mutant cell lines had no detectable GPI enzymatic activity, suppressed completely lactic acid secretion and grew by reprogramming their bioenergetic metabolism to OXPHOS (39). Surprisingly, in contrast to previous pharmacological inhibition studies (29, 37), GPI-KO cells growth was only reduced by twofold in normoxia with ATP produced by OXPHOS being sufficient to maintain their growth and viability. However, the growth rate of GPI-KO cells was severely reduced in hypoxia (1% O₂) while cells remained viable. Interruption of the glycolytic flow by GPI-KO increases the intracellular G6P pool, which in turn was proposed to elicit a short-term inhibition of HK and a long-term inhibition of glucose transport (40, 41). Indeed, we found that both GPI-KO cell lines had decreased GLUT1 expression, as well as induction of thioredoxin-interacting protein expression, a strong negative regulator of glucose uptake (42). We showed that increased OXPHOS dependence of GPI-KO cells made them extremely sensitive to inhibitors of the respiratory chain complexes, such as phenformin and oligomycin (39), in line with the findings of Pusapati et al. (37). Therefore, we speculate that pharmacological inhibition of tumor growth at the level of GPI was effective mainly because of the multiple targets of 2-DG.

In conclusion, we showed that complete suppression of glycolysis in two aggressive cancer cell lines slowed, but did not prevent in vivo tumor growth, in line with the findings of Pouysségur et al. (40) and Pusapati et al. (37). Particularly striking is the LS174T cell line that is highly glycolytic and almost does not respire under normal conditions and is capable to achieve strong re-activation of OXPHOS when challenged by GPI ablation (Figure 2). Consequently, as shown with inducible shRNAs against GPI, the growth was significantly reduced only in combination with mTORC1 or OXPHOS inhibition (37). This remarkable metabolic plasticity of cancer cells revealed as well on several other cell lines (37) poses a big challenge for anticancer therapies targeting metabolism.

Lactate dehydrogenase [(S)-lactate:NAD⁺ oxidoreductase; EC 1.1.1.27] is a family of NAD⁺-dependent enzymes that catalyze the interconversion between pyruvate and lactate, with concomitant oxidation/reduction of the cofactor (NADH/NAD⁺). LDH is a homo- or hetero-tetramer assembled from two different subunits: M and H, encoded by two separate genes, LDHA (M) and LDHB (H), respectively. A third subunit, LDHC, encoded by a separate LDHC gene, is expressed only in testes and sperm and is probably a duplication of the LDHA gene (43). LDH tetramers form at least six isoenzymes that differ in electrophoretic mobility, Km for pyruvate and lactate, immunological characteristics, thermal stability and inhibition by coenzyme analogs or excess pyruvate (44). The existence of mitochondrial LDH was shown in prostate cancer cells (45), and human hepatocellular carcinoma cells (46). Mitochondrial metabolism of lactate results in export of oxaloacetate, malate, and citrate outside mitochondria, therefore having an anaplerotic role (Figure 1) (46). In this mini-review, we will focus on the cytosolic LDH and refer readers to excellent reviews on this topic (47, 48).

Lactic acid, the end product of fermentative glycolysis abundantly released by cancer cells, has a strong impact in tumor microenvironment (72, 73). It can function as an oxidizable fuel, gluconeogenetic precursor and a source of TCA cycle intermediates (46, 74, 75). In addition, it is an antioxidant promoting angiogenesis, migration (76), and its contribution to tumor acidosis was reported to blunt tumor-immune response by T and NK cells (60). Lactic acid is exported/imported in cells by a family of four reversible MCTs [for review, see Ref. (77)]. MCTs as H⁺/Lactate⁻ symporters facilitate net lactic acid exchange across the plasma membrane, whose direction depends on the concentration gradients of protons and monocarboxylate (77). Increasing experimental evidences support the cell–cell and intracellular lactate shuttles hypothesis proposed by Brooks (48), thus lactate is continuously formed and consumed in different cells under fully aerobic physiological conditions (48). MCT1 facilitates lactate and pyruvate transport, it is induced by c-Myc and expressed virtually in all cells. In contrast, MCT4 is an efficient lactate exporter induced by hypoxia and expressed in glycolytic tissues and cancer cells (77). Both MCT1 and 4 need assistance from the chaperone CD147 or basigine (BSG) to express active transporters at the plasma membrane.

Several reports from Baltazar’s group (78–80) have shown that increased expression of MCT1 and MCT4 are associated with a poor prognosis in several types of human cancer, such as neuroblastoma, colorectal carcinoma, gastrointestinal stromal tumors, and prostate cancer. In parallel, our group, exploring pHi-regulating systems as putative anticancer targets in hypoxic tumors (81, 82), developed an interest in blocking lactic acid export. Pharmacological blockage with the specific AstraZeneca MCT1/2 inhibitor (AZD3965) was very efficient in arresting growth of tumors expressing only MCT1, like in transformed fibroblasts (83) or neoplastic B cells (84). However, it became clear that most aggressive cancers express both isoforms, like in colon adenocarcinoma, glioblastoma or non-small cell lung cancer. In these cancer types, genetic disruption of the chaperone (BSG), with zinc finger nucleases, reduced lactic acid export by 70–80%, an action sufficient to re-activate OXPHOS and maintain tumor growth (85). These tumor cells behaved like GPI-KO or LDHA/B-DKO with growth arrest and loss of cell viability induced by inhibitors of mitochondrial respiration (85, 86). However, pharmacological inhibition of MCT1 combined with a MCT4-KO was able to slow considerably in vitro growth and in vivo tumor xenografts (85, 86). We also confirmed that dual pharmacological inhibition of MCT1 and MCT4 considerably reduced cell growth. Removal of the inhibitors after a week allowed cells to form colonies, indicating a cytostatic, not cytotoxic effect induced by lactic acid sequestration in response to MCTs blockade.

Comparing the three independent approaches of interrupting the glycolytic flux, we reach a common consensus and a strong divergence. Genetic disruption of GPI, LDHA/B, or MCT1/4 leads to re-activation of OXPHOS with tumor growth maintenance but increased sensitivity to mitochondrial inhibitors. The case of MCT1/MCT4 is interesting because the phenotype depends on the value of MCT suppression. Partial MCT suppression reached in BSG-KO cells, growth is maintained; total block with dual inhibition by AZD compounds, growth is compromised due to intracellular acidification.

Finally, targeting tumor metabolism via anti-glycolytic thera-pies remains an attractive therapeutic approach (82, 87), especially in combination with the inhibition of mitochondrial pathways, but it will have to be precisely administered in order to spare normal cells and limit toxicity (82).

MŽ isolated and characterized LDHA and B mutant cells, IM isolated and characterized MCT and BSG mutant cells and MP isolated and characterized GPI-mutant cells. SP helped with manuscript editing. MŽ and JP designed the project and wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.