Autonomous glucose metabolic reprogramming of tumour cells under hypoxia: opportunities for targeted therapy

Three members of the human HIF family have been identified: HIF-1, HIF-2, and HIF-3. These heterodimers are composed of α and β subunits, which dissociate under normal oxygen conditions [19]. HIF-1 and HIF-2 are the main transcription factors involved in cell adaptation to hypoxia [20]. HIF binds to the hypoxia response element (HRE) in the promoter region of the target and is involved in tumour cell survival, angiogenesis, glycolysis, and invasion/metastasis. HIF is a heterodimer consisting of a HIFα protein subunit expressed only during hypoxia and a constitutively expressed HIF-1β protein subunit. Under normoxic conditions, oxygen-dependent hydroxylation leads to the recognition of HIFα by the von Hippel-Lindau (pVHL) tumour suppressor, which recruits the E3 ubiquitin ligase, resulting in ubiquitination and protease degradation of HIFα [21, 22]. This prolyl-4-hydroxylase (PHD)-catalysed hydroxylation reaction is coupled with the oxidative decarboxylation of 2-oxoglutarate (2-OG), and succinate and carbon dioxide are produced [21]. Notably, all three PHD enzymes can utilize O₂ as substrate and 2-OG, Fe²⁺ and ascorbic acid as co-substrates to hydroxylate both HIF-1α and HIF-2α. Compared to the loss of PDH2 heterozygosity or homozygous PDH3, a single lost PHD1 allele has the ability to induce hypoxia tolerance in mice, thus highlighting the functional specificity of PHD in vivo [23‐25]. In vivo experiments, compared to the loss of heterozygous PDH2 or homozygous PDH3, a single lost mouse carrying PHD1 has the ability to induce hypoxia tolerance, thus highlighting the functional specificity of PHD in vivo [26]. Under hypoxia, the hydroxylation of HIFα is inhibited, leading to the stabilization of HIFα. HIFα dimerizes with HIF-1β to form a transcriptional activation complex. In addition, HIF asparaginyl hydroxylase or factor inhibiting HIF-1(FIH-1), a member of the Fe²⁺ and 2-OG-dependent dioxygenase family, can inhibit the transcriptional activity of HIFs by targeting the C-terminal transcription activation domain (CTAD) of HIF1-α and HIF-2α under normoxia to block the interaction between HIFα, p300 and CBP [27]. Importantly, due to the differences in the amino acid sequence of the protein subunits of the HIFα adjacent to the hydroxylated asparagine residue, the efficiency of CTAD hydroxylation of HIF-2α is lower than it is for HIF-1α [21, 28, 29]. Another difference is that HIF-1α expression is ubiquitous, while HIF-2α expression is restricted to specific tissues [30]. Furthermore, the heterogeneity of HIF protein isomers is also manifested by the lack of CTAD action with HIF-3α, which results in alternative splicing of HIF-3α to form an inhibitory PAS domain-containing protein, forming a transcriptionally inactive heterodimer with HIF-1α to inhibit the HIF reaction [19]. In addition to HIFα, FIH-1 has other substrates, the physiological functions of which need to be further explored [31, 32] (Fig. 1). In addition, some other potential targets of PHD, such as molecular scaffolds, may require structures to be effectively hydroxylated in tumour cells.

PHD is known to promote the hydroxylation reaction of HIFα, thereby inducing the degradation of HIFα. Current research has revealed many proteins that can regulate the stability of HIF-1α. For example, overexpression of adenylate kinase 4 (AK4) promotes the stabilization of HIF-1α by increasing intracellular ROS levels, subsequently inducing the epithelial-mesenchymal transition (EMT) and enhancing tumour invasion potential under hypoxic conditions [33]; BRCA1-IRIS is a chromatin-associated replication and transcriptional regulator that is overexpressed in a variety of primary human cancers and can prevent glycogen synthase kinase-3 (GSK-3β) from recruiting F-box protein (Fbw7), a tumor suppressor associated with chromosomal instability and some types of malignancy, through phosphorylation of HIF. Fbw7 can further mediate ubiquitylation and degradation of HIF [34, 35]; Collagen prolyl 4-hydroxylase (P4H) is an essential catalytic enzyme in the progression of breast cancer. The α1 subunit of P4H (P4HA1) regulates the levels of 2-OG and succinic acid, suppressing the hydroxylation of proline in HIF-1α, thereby enhancing HIFα stability in cancer cells [36]. Additionally, HIF-induced proteins can also inhibit the degradation of HIFα through a feedback loop. The overexpression of HIF-2α in pancreatic cancer cells leads to the nuclear translocation of β-catenin, and the formation of the HIF-2α/β-catenin complex significantly prolongs the half-life of HIF-2α, maintaining the stability of HIF-2α [37]. These recently discovered proteins can be targeted to inhibit the stability of HIFα, thereby regulating tumor cell growth. Most of the proteins discovered in recent studies are described in detail in Table 1.

The importance of HIF in the microenvironment has made increasing researchers pay attention to the stability of HIF. However, current research has not fully explained the interaction mechanism between HIF and other molecules. For example, a recent study found that in hypoxic TME, enriched miR-301a-3p could be transmitted between GC cells via exosomes and then contributed to inhibit HIF-1α degradation through targeting PHD3 [45]. This means that extracellular vesicles (EVs) with proteins or RNA can regulate the stability of HIF in tumor cells. It has been widely recognized that HIF regulates the secretion of EVs under hypoxic conditions [46]. However, there are few studies on the negative feedback effect of EVs on the stability of HIF, which needs more in-depth research to explain the mechanism.

Glucose catabolism mainly includes the anaerobic oxidation of sugar, aerobic oxidation of sugar and the pentose phosphate pathway, depending on the metabolic characteristics and oxygen supply status of different types of cells [47]. The Warburg effect allows tumour cells to gain survival advantages in two ways. One is to increase carbon sources, which are used to synthesize proteins, lipids, and nucleic acids to meet the needs of tumour growth; the other is to turn off aerobic oxidation channels to prevent the generation of free radicals, thereby preventing apoptosis [12]. Therefore, under hypoxic conditions, HIFα can regulate tumour growth by regulating the anaerobic and aerobic oxidation of glucose.

In addition to regulating glucose catabolism, HIF can increase cancer glycogen reserves [61] under hypoxia. Pelletier et al. found that the glycogen content of cancer cell lines cultured in 1% oxygen increased from 5 to 37-fold, indicating that the synthesis of glycogen is HIF-1α-dependent. Within 24 h of glucose removal from the culture medium, cells that accumulate glycogen under hypoxic conditions have higher viability (70–80%) than cells cultured under normoxic conditions (20–60%) [62]. In addition, PHD2 gene inactivation or systemic pharmacological inhibition in neutrophils can lead to the stabilization of non-hypoxic HIF-1α, thereby enhancing glycogen storage. Therefore, it is necessary to understand how HIF regulates glycogen metabolism. Glycogen originates from the allosteric formation of glucose-6-phosphate (G6P), glucose-1-phosphate (G1P) and uridine triphosphate (UTP) to form uridine diphosphate glucose (UDPG) [61]. UDPG is connected by α-1,4-glycosidic bonds to form a straight chain and connected by α-1,6-glycosidic bonds to form branches, and these formed branches can increase the water solubility of glycogen.

The synthesis and decomposition of glycogen involve the activities of various enzymes and regulatory proteins. Among these, glycogen synthase (GS) and glycogen phosphorylase (GP) are key enzymes in the process of catalysing glycogen synthesis and degradation, respectively. GS elongates the glycogen branch by forming an α-1,4 glycosidic bridge, while GP cleaves these glycogens, releasing G1P, which enters the glycolysis pathway, or G6P that enters the PPP [63]. This result suggests that the pathways by which HIF regulates glycogen metabolism include the key regulatory enzymes and related kinases of glycogen synthesis and decomposition. Studies have shown that under hypoxic conditions, HIF not only upregulates the expression of UDP-glucose pyrophosphorylase and promotes glucose activation before glycogen synthesis but also inhibits liver glycogen phosphorylase (PYGL) by promoting glycogen synthase 1 (GYS1) regulation of glycogen synthesis. Moreover, the activation of glycogen branching enzyme (GBE1) by HIF can further regulate the formation of glycogen branches [56, 63]. Interestingly, direct regulation of key enzymes is not the only mechanism by which glycogen metabolism is modulated. In fact, indirect effects on key enzymes can also regulate glycogen synthesis. For example, the HIF-1α-dependent expression of PP1 complex phosphatase (PPP1R3C) in human MCF7 cells activates GYS1 while reducing the breakdown of glycogen into glucose monomers [64]. These HIF-dependent effects allow glycogen to accumulate under conditions of hypoxia and nutritional deficiencies, affecting cell responses.

Mitochondria, organelles that generate energy in the body, are composed of simple organophospholipid bilayer membranes, intermembrane spaces, complex internal phospholipid bilayers, and mitochondrial matrices. They are especially enriched in the heart muscle, skeletal muscle, liver, kidney, and especially neuronal cells, which need high levels of energy to function [65]. The biological oxidation of glucose is a precise process that relies on the oxidized the mitochondrial respiratory chain to transfer electrons from NADH and succinate to oxygen and finally the combination of hydrogen protons and oxygen to form water and release ATP [66]. The oxidized respiratory chain consists of four mitochondrial complexes located on the inner mitochondrial membrane, called complexes I, II, III, and IV. They are NADH-CoQ reductase, succinate-CoQ reductase, CoQ-cytochrome c reductase, and cytochrome c oxidase. Ubiquinone (CoQ) and cytochrome c are two freely diffusible molecules that mediate electron transfer between complexes [67]. Current research shows that HIF can regulate the biological oxidation of glucose through the regulation of mitochondria in a hypoxic environment, including the regulation of mitochondrial biogenesis, the generation of ROS [68, 69] and the removal of mitochondria.