Progress of research on glucose transporter proteins in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a malignant tumour with high prevalence and mortality rate worldwide. Metabolic reprogramming of cancer cells may be a major factor in the process of this disease. Glucose transporter proteins (GLUTs) are members of the major facilitator superfamily of membrane transporters, playing a pivotal role in the metabolic reprogramming and tumour progression in HCC. This review discusses the advances in the study of GLUTs in HCC, including the expression patterns, functions and possibilities of GLUTs. In HCC, the expression levels of GLUTs are closely associated with tumour aggressiveness, metabolic reprogramming and prognosis. A series of inhibitors have been demonstrated efficacy in inhibiting HCC cell growth and glucose uptake in in vitro and in vivo models. These inhibitors offer a novel approach to HCC treatment by reducing the glucose metabolism of tumour cells, thereby impeding tumour growth, and concurrently enhancing the sensitivity to chemotherapeutic agents. This reminds us of the urgent need to elucidate GLUTs’ roles in HCC and to determine the most effective ways to translate these findings into clinical practice.

Key Words: Hepatocellular carcinoma; Glucose transporter protein; Metabolic reprogramming; Therapeutic target; Drug resistance

Core Tip: Hepatocellular carcinoma (HCC), a predominant liver cancer subtype, exhibits a high mortality rate mainly due to difficulties in early detection and rapid disease advancement. Glucose transporter proteins, essential for glucose uptake, significantly influence HCC progression and are pivotal in glucose metabolism and tumor development.

INTRODUCTION

Hepatocellular carcinoma (HCC) is a major subtype of liver cancer, a third leading cause of cancer death worldwide[1]. Despite some progress in the diagnosis and treatment of HCC, patients are facing a poor prognosis, due to the difficulty of diagnosis at the earlier stages, and high prevalence of tumour invasion, metastasis and recurrence. In addition to infections of hepatitis B virus (HBV) and hepatitis C virus (HCV)[2], the incidence of HCC is also related to the presence of fatty liver and alcoholic cirrhosis resulted from unhealthy dietary and smoking habits, obesity problems, diabetes and excessive accumulation of iron in the body[3]. These factors pose a threat for an increasing number of patients with HCC. Therefore, to realize early diagnosis and find promising therapeutic targets for HCC, more specific biomarkers are required to elaborate the carcinogenesis and progression of HCC.

MECHANISM OF TUMOUR CELLS

The liver is a major site of carbohydrate synthesis, storage and redistribution to remain metabolic carbohydrate homeostasis, and hepatocytes are the major cells involved in glucose regulation and glycogen storage[4]. Considering the significance of molecular regulation of hepatic glucose homeostasis, it highlights an intimate association of abnormal glucose metabolism with the development of HCC. Glucose transporter proteins (GLUTs), as key transporters mediating glucose entry intracellularly, occupy a key position in glucose metabolism and hence HCC progression. Therefore, studying the expression and function of GLUTs in HCC may provide explanatory evidence to interpret HCC pathogenesis and to develop new therapeutic strategies.

Metabolic reprogramming and the Warburg effect in tumour cells

The tumor microenvironment (TME) is characterized by low oxygen, acidity, and high osmotic pressure, conditions that tumors rely on for survival. To adapt to hypoxia and nutrient deficiencies[5], tumor cells adjust their energy metabolism to generate the biological energy required for rapid growth and proliferation. This adaptation also meets the biosynthesis and oxidation-reduction reaction (REDOX) needs of the cells. This phenomenon, known as metabolic reprogramming, is a key feature of cancer[6]. It is prevalent in nearly all tumorigenesis and developmental processes and is strongly associated with tumor cell transformation, invasion, and metastasis[7]. Metabolic reprogramming is mediated by various factors, including changes in oncogenes, tumor suppressor genes, growth factors, and tumor-host cell interactions. These factors help cancer cells meet their anabolic needs[8], promote tumor growth, and regulate metabolism by altering the expression and activity of key metabolic enzymes. Oncogenes, which are mutated or overexpressed forms of proto-oncogenes, drive uncontrolled cell growth and proliferation. They are closely linked to metabolic changes in cancer cells[9]. The imbalance in oncogene-driven metabolic pathways gives tumor cells a selective advantage, enabling them to proliferate rapidly and survive in hostile microenvironments. The imbalance in the cell division cycle and rapid proliferation of cancer cells depend not only on genetic changes but also on energy sources and metabolic shifts[10]. The recognition of energy’s importance has led to the resurgence of cancer metabolism as a field closely related to tumor genetics. Cells primarily obtain energy through glucose metabolism, which includes pathways such as the tricarboxylic acid cycle, glycolysis, and pentose phosphate pathway. Cancer cells consistently generate energy through aerobic glycolysis. While glycolysis is a physiological response to hypoxia in normal tissues, Warburg[11] observed in the 1920s that tumors also exhibit high rates of glucose uptake and lactate production under aerobic conditions (the Warburg effect)[12]. Although glycolysis is less efficient at producing adenosine triphosphate (ATP), it is faster than oxidative phosphorylation. This rapid ATP production is thought to contribute to the fast proliferation of cancer cells[13]. It is now understood that the metabolic reprogramming behind the Warburg effect is driven by several oncogenes and tumor suppressors. To maintain enhanced proliferation, cancer cells have an increased demand for glucose, fatty acids, and amino acids, which serve as both energy sources and building blocks for macromolecules. Although cancer cells alter the metabolism of all nutrient substrates, carbohydrate metabolism has garnered particular attention because cancer cells rely heavily on glucose metabolism to produce most of their energy[14]. Increased glucose transport in malignant cells is associated with the upregulation and dysregulated expression of glucose transporters. Other findings involving cell transformation support this link[15] (Figure 1).

Figure 1 The process of glucose metabolism. Glucose enters the cell through glucose transporter proteins (GLUTs). Glucose from the external environment is transported into the cell via the GLUT transporter. In the cytoplasm, glucose is converted to glucose-6-phosphate (G6P) by hexokinase. G6P is then converted to fructose-6-phosphate (F6P) by phosphor glucose isomerase. Phosphofructokinase-1 converts F6P to fructose-1,6-bisphosphate (FBP). FBP is split by aldolase into dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). Triose phosphate isomerase converts DHAP to G3P. G3P is oxidized by glyceraldehyde-3-phosphate dehydrogenase to 1,3-bisphosphoglycerate (1,3-BPG), producing nicotinamide adenine dinucleotide in the process. 1,3-BPG is converted to 3-phosphoglycerate (3-PG) by phosphoglycerate kinase. 3-PG is isomerized to 2-phosphoglycerate (2-PG) by phosphoglycerate mutase. 2-PG is converted to phosphoenolpyruvate (PEP) by enolase. The final step is catalyzed by pyruvate kinase, which converts PEP to pyruvate. Pyruvate then enters the mitochondria for oxidative phosphorylation and proceeds through the tricarboxylic acid cycle to produce adenosine triphosphate. GLUT: Glucose transporter proteins; HK: Hexokinase; PGI: Phosphor glucose isomerase; G6p: Glucose-6-phosphate; F6P: Fructose-6-phosphate; PFK1: Phosphofructokinase-1; FBP: Fructose-1,6-bisphosphate; DHAP: Dihydroxyacetone phosphate; TPI: Triose phosphate isomerase; G3P: Glyceraldehyde-3-phosphate; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; 1,3-BGP: 1,3-bisphosphoglycerate; 3PG: 3-phosphoglycerate; PGK: Phosphoglycerate kinase; PGM: Phosphoglycerate mutase; 2PG: 2-phosphoglycerate; PK: Pyruvate kinase; PEP: Phosphoenolpyruvate; TCA: Tricarboxylic acid cycle; ATP: Adenosine triphosphate.

Important regulators in the Warburg effect: GLUTs

As for the primary function of glycolysis, it can promote glucose consuming and converting into energy in a form of ATP. Due to its lipophilic nature, the cell membrane is impermeable to glucose and requires specific carrier proteins for its transport. The family of GLUTs, encoded by solute carrier family 2 members (SLC2A), comprises 14 members that generally act as carrier proteins to facilitate transmembrane transport. It allows for the entry of extracellular glucose into the cell without the expenditure of energy[16].

GLUTs are members of the “sugar porter (SP)” family within the “major facilitator superfamily”. They are composed of 12 transmembrane helices (M1-M12), divided into N-domains (M1-M6) and C-domains (M7-M12), displaying pseudo-double symmetry. An intracellular helix connects these two domains (ICH). The N and C termini of all GLUT proteins are located in the cytoplasm. Within the cell, the N- and C-domains contain characteristic sequences of the SP family, which are implicated in the transport and regulation of intracellular energy. GLUTs undergo various conformational states during transport: Outward opening for binding extracellular substrates; Outward occluded, wherein conformation changes post-substrate binding to prepare for transport; and Inward-open for releasing substrates into the cell[17,18]. GLUTs facilitate the passive entry of extracellular glucose into cells by alternating conformational states, thus promoting diffusion along the concentration gradient without expending energy[16]. All these members, with identified expressions in humans[19] are crucial for facilitating glucose uptake by cancer cells. In cancer cells, GLUT expression is increased to support enhanced glycolytic processes to meet the high demand for energy and biosynthetic precursors in cancer cells. Based on sequence similarity, GLUTs can be classified into the following distinct classes[20,21]: Class I consists of the widely characterized glucose transport proteins GLUT1 GLUT4 and GLUT14, which are distributed in different tissues (GLUT1: Erythrocytes, cerebral microvessels; GLUT2: Liver, pancreatic islets; GLUT3: Neuronal cells; GLUT4: Muscle, adipose tissue) and are involved in hormonal regulation (GLUT4: Insulin sensitivity). Class II consists of GLUT5 (a fructose-specific transporter) and three related proteins GLUT7, GLUT9 and GLUT11. Among them, there has been data related to the fructose-inhibitory glucose transport activity of GLUT11 in a reconstituted vesicular system[22]. Class III (GLUT6, 8, 10, 12 and the inositol transporter protein HMIT1)[23,24]. With the exception of GLUT7, GLUT13 and GLUT14, the rest of the class is expressed in liver tissue. High levels of glucose metabolism are observed in HCC. In HCC, the expression patterns of GLUT1, GLUT2 and GLUT3 differ from those of normal hepatocytes, which may be related to the metabolic properties of HCC and the biological behaviour of the tumours[25]. This review will focus on glucose transport proteins that are closely associated with HCC.

GLUTs participate in multiple disease processes and are regulated by multiple factors

GLUTs are implicated in various diseases, including metabolic disorders, cardiovascular diseases, neurodegenerative conditions, and cancer. GLUT2, primarily expressed in the liver, is crucial for glucose uptake and release[26]. In conditions like nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, GLUTs may play a role in liver metabolic reprogramming. In type 2 diabetes, reduced GLUT4 expression in muscle and adipose tissues leads to decreased glucose uptake, impairing blood sugar regulation[26]. GLUTs also facilitate glucose metabolism in the heart and blood vessels, influencing cardiovascular health. For example, GLUT4 is expressed in cardiomyocytes and regulates cardiac energy metabolism[27]; GLUTs regulate glucose uptake and energy supply in neurons, with GLUT3 specifically involved in brain energy metabolism[28]. GLUT9 influences uric acid metabolism, and its dysfunction may result in hyperuricemia and gout[29]. Additionally, GLUTs regulate the metabolism of amino acids, lipids, and other small molecules[30,31]. Therefore, GLUTs are implicated in the progression of various benign and malignant diseases across different tissues. Variability in GLUT expression across cell types and tissues can be attributed to factors such as transcription factors, microRNAs, and epigenetic changes, including DNA methylation[32] and histone modifications[33]. Post-translational modifications, such as phosphorylation[34] and glycosylation, may alter GLUT stability, localization, and activity. Intracellular signaling pathways, including adenosine 5’-monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), and mitogen-activated protein kinase activation, may modify GLUT function by affecting their expression or activity[35,36]; cellular environmental factors such as intracellular potential of hydrogen (pH), REDOX state, and energy state (e.g., ATP/adenosine diphosphate ratio)[30] may influence GLUT conformation and activity. Moreover, the extracellular microenvironment, including changes in hypoxia[37], acidosis[38], inflammatory factors, extracellular matrix components, and hormone levels in the TME, may impact GLUT function[39]. Understanding these factors is essential to elucidate GLUTs’ roles in both physiological and pathological conditions and to aid in developing targeted therapies and treatment strategies.

GLUTs are involved in HCC on the basis of their roles in cancer cells

The active consumption of glucose relies generally on specific transporter proteins, known as GLUTs[9], to activate the movement of glucose across cellular membranes. These proteins are usually overexpressed in almost all types of malignancies. Increased uptake of glucose by GLUTs provides a greater substrate for this effect, resulting in elevated lactate production in tumour cells[40]. Lactate production can further lead to a reduction in the pH of the TME. This, in turn, disturbs the tumour immune microenvironment, inhibiting immune cell activity and promoting tumour immune escape. Consequently, there may be progression of the tumour and its subsequent invasion and metastasis. GLUTs are regarded as prospective therapeutic targets in cancer given their pivotal function in tumour cell metabolism. Elevated expression of GLUTs has been observed in circumstance of tumour resistance to chemotherapeutic agents. For example, the association of elevated GLUT1 expression with chemotherapeutic drug resistance has been observed in a number of different cancers. This phenomenon may be attributed to the fact that tumour cells evade the lethal effects of chemotherapeutic agents by enhancing glucose uptake to sustain their energy demands. Rather than a sole implication in glucose uptake; GLUTs may also influence the metabolic reprogramming of tumour cells, including the modulation of mitochondrial function and oxidative phosphorylation. It consequently impacts tumour cell energy metabolism and biosynthetic pathways. The high metabolic demands of cancer cells necessitate a significant uptake of glucose, serving as the foundation for diagnostic techniques. One of the representatives is a non-invasive diagnostic and prognostic technique of fludeoxyglucose (18F-FDG)-positron emission computed tomography (PET), with the use of 18F-FDG[41] containing non-metabolic analogue (2-deoxy-D-glucose) of glucose. A multitude of glucose transporters (GLUTs) on the hepatocytes enable 18F-FDG transporting from the blood into hepatocytes. The tracer 18F-FDG is distributed on the plasma membrane of hepatocytes and phosphorylated to 18F-FDG-6-phosphate by hexokinase (HK). Thus, a higher uptake rate of 18F-FDG may indicate a stronger aggression and advanced stages of tumours[42]. This diagnostic technique is particularly widely employed within the field of oncology. Therefore, high GLUT expression renders 18F-FDG-PET a highly effective diagnostic tool. In a study of the association of GLUT1-5 with FDG uptake, significantly elevated GLUT1 and GLUT3 expressions contribute to the accumulation of FDG in malignant tumours[43]. Another investigation of 22 different cancers demonstrated an association of GLUT1 and GLUT3 in HCC[44], and several other cancers[45]. Moreover, differential expression of GLUTs was noticed in two subgroups of high standardized uptake value (SUV) max (SUV max > 8) and low SUV max (SUV max < 5)[44]. In particular, in patients with high SUV max, overexpression of the two proteins was related to high glucose uptake and resulted in a more aggressive HCC phenotype, including larger tumours, a high recurrence rate and poor survival[45].

The liver is a major organ with a close association with glycogen metabolism. Alterations in GLUT expression may be more pronounced in tumours that originate in the liver. The Warburg effect is a characteristic feature of glucose metabolism in HCC, providing a metabolic foundation for rapid HCC cell growth and survival. This metabolic shift is dependent on the aberrant expression of various transport proteins and catalases. In HCC, complex roles have been uncovered concerning GLUT1, GLUT2, GLUT3, GLUT5, GLUT6, and GLUT9[46]. GLUT1 in HCC[47] is overexpressed to promote aerobic glycolysis in tumour cells, which is associated with tumourigenesis and progression. In HCC, GLUT1 showed altered expression on the tumour endothelium, serving as an important prognostic and diagnostic marker. Given that GLUT1 is not expressed in normal hepatocytes and peritumoural biliary epithelium, it may be employed as a marker to differentiate between cholangiocarcinoma and HCC[1,48]. Despite elevated expression in HCC[49], GLUT2 had downregulated expression in preneoplastic and neoplastic liver disorders[14]. Higher GLUT3 expression in HCC is associated with increased alpha-fetoprotein levels, larger tumour size, poor histological differentiation, as well as poorer prognostic and diagnostic properties. GLUT5, GLUT6 and GLUT9 are also reported in the progression of HCC. For instance, GLUT5 expression was markedly elevated in liver metastatic lesions relative to primary lung tumours and was also increased in HCC[50,51], illustrating its significance in predicting HCC metastasis. GLUT6 was identified solely in HCC cell line[25] whereas GLUT9 expression was observed in the cytoplasm of perihepatic cells surrounding the core of HCC. The distinct roles of GLUTs in HCC are underscored based on the above studies, offering insights into potential avenues for HCC research and management.

Hypoxia inducible factor-1α enhances the role of GLUTs under hypoxic conditions

The TME is composed of tumour cell and their organelles-released growth factors and cytokines, which together create an ecological environment that may be advantageous to cancer cells under unfavorable hypoxia, nutrient deficiencies, and necrosis[52]. Tumour hypoxia is an important microenvironmental factor inducing canceration and anti-tumour resistance. Adaptation to a hypoxic environment is a fundamental process for cancer cell survival and growth[53]. The abnormal and dysfunctional tumour vasculature is unable to restore oxygenation due to a loss of oxygen transport, which results in the persistence of hypoxia within the TME. This, in turn, promotes cancer cell migration, invasion, immune escape and drug resistance. As a consequence of increased glucose depletion, glucose deprivation and hypoxia were induced in the TME of HCC cells, accumulating hypoxia inducible factor (HIF)-1α and activated key enzymes involved in glycolysis finally[54]. This process drives cancer progression. In conditions of hypoxia that result in drug resistance and a poor prognosis for HCC, HIF-1α mediates a metabolic switch that blocks pyruvate-acetyl coenzyme A conversion and inhibits lipid oxidation, thereby promoting cancer cell survival and proliferation[55]. In mouse HCC cells, a GLUT1 promoter-bound HIF-1α/ARNT complex increased GLUT1 expression. It also resulted in intracellular compartment translocation of GLUT1-containing vesicles to the cell membrane. In human HepG2 cells, the proposed complex could also regulate the expression of GLUT1 and GLUT3 by binding to their promoter regions[56].

High GLUT1 expression is associated with poor prognosis of HCC

In general, GLUT1 has low or undetectable expression in normal and benign liver tissue[44,57], but is abundantly expressed in HCC cells, suggesting its crucial role in glucose uptake by HCC cells. Concerning its direct effect in HCC tumourigenicity, inhibition of GLUT1 expression would retard HCC cells to proliferate and migrate. The significance of GLUT1 in HCC is regulated by a variety of mechanisms and signalling pathways and can be coupled with a variety of methods to inhibit expression in HCC cells. For example, GLUT1 was effective in the treatment of HCC by overexpression of the cell line (Huh7 and HepG2) through miR-455-5p, which researchers have found to relate to impaired cell proliferation, colony formation, migration and invasion[58]. Insulin-like growth factor-1 receptor (IGF-1R) can increase GLUT1 expression by activating AKT signalling to accelerate glycolysis in cancer cells. Through suppression of IGF-1R expression by miR-455-5p, it would reduce AKT phosphorylation and GLUT1 expression. The impairment of these functions is associated with the regulation of miR-455-5p through IGF-1R/AKT/GLUT1 axis. GLUT1 expression could also be modulated by affecting nuclear factor kappa-B (NF-κB) in the -1008/-780 region of the GLUT1 promoter. Thus, the use of pyrrolidinedithiocarbamate ammonium (an NF-κB inhibitor) was able to inhibit GLUT1 expression, which in turn affected reactive oxygen species levels and glucose consumption.

FOXM1, with high expression in several solid tumours, is involved in cell proliferation, cell cycle progression, and epithelial-mesenchymal transition. In HCC, FOXM1 acts as a promoter of glycolysis and results in poor prognosis[59]. While for intervention, various HCC cell lines, the expression of FOXM1 was knocked down, which downregulated GLUT1 and hence positively regulated glycolysis[60]. The FOXM1-GLUT1 axis may be a therapeutic target for HCC. A published research explored SLC2A1-AS1, an unannotated antisense long non-coding RNA, in HCC. By suppressing STAT3/FOXM1/GLUT1, it inhibited aerobic glycolysis and HCC progression, highlighting its value for preventing HCC recurrence[61]. In another study by Shang et al[62], knockdown of RRAD in the MHCC-97H and HepG2 cell lines upregulated GLUT1 mRNA levels obviously. It supports the inhibitory role of RRAD in aerobic glycolysis in HCC, and its mechanism may be related to a negative modulation of GLUT1 and HK-II. It would mediate glycolysis negatively in HCC cells, supported by the enhancement of glucose utilisation and lactate production when the expression of RRAD was suppressed.

High expression of GLUT2 in hepatocytes may be associated with good prognosis

With expression detected mainly in liver and pancreatic islet cells, GLUT2 also acts as a glucose sensor to regulate secretion and gluconeogenesis processes, in addition to a participator of glucose transport. GLUT2 can stimulate metabolism and release metabolites related to glucose-sensitive gene transcription, largely depending on its low affinity and high capacity[63]. Notably, GLUT2 exhibited greater prognostic value in patients without major risk factors (e.g., alcohol consumption and chronic HBV/HCV infections)[49]. An analysis on GLUT2 protein expression and clinical stage showed a negative correlation between GLUT2 protein expression and liver malignancy[64]. Moreover, high expression of GLUT2 appeared to indicate a favourable prognosis in individuals who consumed alcohol or were infected with viral hepatitis, despite none obvious relationship with overall survival. Although previous findings have shown both the diagnostic and prognostic values of GLUT2 for HCC. However, no association between this factor and HCC patients’ overall survival was generated in a recent meta-analysis with limited studies involving less samples[65].

GLUT3 is highly expressed and is associated with drug resistance in HCC

GLUT3 is detected with high expression specifically by neurons with unique properties suited to cell-specific expression and function[66]. As an adaptive response, its regulation can prevent cellular damage in case of reduced cerebral metabolic energy. GLUT3, carrying glucose intracellularly, maintains cellular glucose metabolism and participates in the process of tumourigenesis. Tumour patients with higher GLUT3 expression may have lower survival[67].

A study by Gao et al[68] explored the expression of GLUT3 in HCC, and its elevated level was associated with diminished overall survival in HCC patients following tumour resection. Besides an indicator of serum alpha fetoprotein levels, GLUT3 overexpression might also suggest large tumour size, poor differentiation and higher (III-IV) tumor node metastasis stages. It may be a useful biomarker for monitoring HCC patients’ survival. It has been found[67] that in the presence of GLUT3 overexpression, tumour cells shared the same metabolic phenotype with bevacizumab-resistant cells, conferring drug-resistant tumours with rapid growth and bevacizumab-resistant response. Therefore, therapeutic agents targeting the blockage of GLUT3 can be exploited to improve immunotherapy-resistant HCC patients’ prognosis.

Intra-hepatic iron accumulation is frequently observed in pathological conditions (e.g. cirrhosis and cancer). Increased expression of GLUT1 and GLUT3 in patients with HCC correlates with reduced overall survival. Ribeiro et al[69] discovered that iron loading increased intra-hepatic GLUT3 expression through LKB1/AMPK/CREB1 signalling axis to regulate cell proliferation in HCC. GLUT3 expression. Iron overload promoted cell proliferation in HCC by increasing GLUT3 expression, supporting that inhibition of GLUT3 or reduction of iron levels may provide new therapeutic strategies for HCC.

HBV, HCV and GLUTs in HCC

Particular attention has been paid to the role of GLUTs in HCV and the HBV, both of which are closely associated with HCC progression[46]. The hepatocytes-invaded DNA virus of HBV is a major cause for global cirrhosis and HCC[70]. HBV infection induces endoplasmic reticulum (ER) stress, particularly through activation of X-box binding protein 1. HIF1 pathway-mediated ER stress can regulate glucose metabolism. The activated HIF1 pathway can lead to increased transcription of GLUT1 and expression of several restrictive glycolytic enzymes that help hepatocytes adapt to the hypoxic environment. Meanwhile, HBV replication is dependent on AMPK-ULK1-induced autophagy signaling, which can be promoted by glucose uptake and glycolysis resulted from activated AMPK-mTOR-ULK1 axis under low glucose levels and inhibition of GLUTs[66,71,72]. While HCV is a single-stranded RNA virus belonging to the Flaviviridae family. Infection with HCV usually lead to advanced liver fibrosis, and cirrhosis, a determinant of precancerous liver cancer. Reports suggest that 80%-90% of HCC cases are caused by cirrhosis[73,74]. By upregulating Ser312 phosphorylation of insulin receptor substrate-1 and promoting its degradation, the core protein of HCV can disturb the transduction of insulin signals to phosphatidylinositol-3-kinase/Akt. HCV proteins interact with ER and mitochondria through interactions that lead to the upregulation of oxidative stress and inflammatory factors (e.g., tumor necrosis factor-α and interleukin-6), which can affect glucose metabolism by influencing GLUT1, GLUT2 and GLUT4 activities[75-77].

In addition to the above common members of GLUTs that play a role in HCC, recent evidence[78] has shown that GLUT4 was highly expressed in HCC. It may be crucial for HCC cell migration and invasion induced by the low-glucose microenvironment and is associated with recurrence. The role of GLUTs in HCC-associated viral infections may provide valuable directions for future research and targets for therapy development. Blockade of potential genetic targets and intervention in the early stages of liver disease may delay liver fibrosis and HCC in viral hepatitis suffers.

GLUT inhibitors as a potential anti-cancer strategy

Cancer cells are dependent on increased glucose use compared to normal healthy cells, suggesting the anti-cancer feasibility of glucose deprivation for cancer prevention. Drug-based inhibition of glucose metabolism alone or with other therapeutic modalities jointly has shown promising anti-cancer activity. A simple and effective approach is to block GLUTs, which would prevent glucose from entering the cancer cell and lead to a complete disruption of the glycolytic pathway. Most inhibitors of GLUTs have been identified in cell-based screening, either directly or indirectly, including natural, natural product-inspired, and non-natural inhibitors targeting GLUTs[79]. At present, experimental drugs targeting GLUTs mainly consist of the following categories: BAY-876, chromopynones, cytochalasin B, DRB18, fasentin, glupin, glutor, rapaglutin A (RgA), STF-31, WZB117 and several other plant-based GLUT inhibitors. GLUT inhibitors of different chemical classes currently include alkaloids, flavonoids and other phenolic compounds[80]. Among these different classes of plant-derived GLUT inhibitors, > 25 compounds have been identified as having anti-tumour activity, such as: Rhizopusin[81], apigenin, curcumin[82] and others. For example, one study demonstrated that capsaicin, extracted from capsicum peppers, suppressed the expression of proteins linked to the Warburg effect, including GLUT1, HK2, pyruvate kinase isozyme type M2, and lactate dehydrogenase, and reduced glucose, lactic acid, and ATP production, thereby significantly increasing the number of terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling-positive apoptotic cells in HCC cells[83]. Genistein, derived from soybeans, inhibits both the expression and activity of HIF-1α, thus blocking GLUT1/HK2 and causing cell cycle arrest at the G2/M phase, while inducing apoptosis and autophagy in HCC cells[84]. Although these GLUT inhibitors show promising anti-tumour potential, the exact mechanism of most of the inhibitory effects remains elusive and should be unravelled for their optimal use in anti-tumour therapy. It is also a great challenge to determine whether the beneficial anti-proliferative effects of these plant-derived compounds on cancer cells are attributed to the blockade of glucose uptake or to their known antioxidant properties[85].

As suggested by an existing research, cytochalasin B could reduce the availability of glucose by competing with substrates of GLUTs for the binding sites to cancer cells, resulting in effective suppression of glucose, deoxyglucose and glucosamine transport, thereby decreasing the glucose supply to cancer cells, without any impact on intracellular phosphorylation and metabolism. This study initially stimulated interest in using glucose metabolism to inhibit cancer tumour growth[86].

Considering that both GLUT1 and GLUT3 are upregulated in various cancers[87], effective inhibition of glucose uptake may require the regulation of GLUT1 and GLUT3 simultaneously[88]. It inspired the development of Glupin and chromopynones, compounds that selectively target GLUT1 and GLUT3 to inhibit cancer cell growth effectively. Glupin is a compound that selectively targets GLUT1 and GLUT3 to inhibit cancer cell growth. Chromopynones are ‘pseudo-natural products’ created by combining conventional natural product NP-derived fragments[89]. The combination of chromane and tetrahydropyran fragments led to the synthesis of a series of 44 structurally diverse chromopynones using the Biginelli multi-component reaction as well as subsequent cyclisation and decarboxylation reactions. These compounds showed selective inhibition of glucose uptake at the same time of targeting GLUT1 and GLUT3 selectively.

Another compound called Glutor shares similar mechanism of action with Glupin[90], which is a nanomolar inhibitor targeting GLUT1, GLUT2 and GLUT3. Glutor effectively induced cell death in multiple cancer cell lines, including HCC, by inhibiting glycolytic processes, with confirmed efficacy in both ex vivo and in vivo cancer cell culture experiments.

In addition to the above inhibitors that target GLUTs alone, there are also inhibitors that work by modulating hormone secretion. RgA is a glucagon-like peptide-1 analogue for type 2 diabetes commonly. It works by stimulating insulin secretion and lowering blood glucose. Data has proposed that RgA[91] could inhibit GLUT1, GLUT3 and GLUT4 potentially. By suppressing glucose uptake, RgA can reduce ATP synthesis intracellularly, activate AMP-dependent kinases, inactivate mTOR signalling, induce cell cycle arrest and apoptosis of cancer cells. In addition, RgA is able to inhibit tumour xenografts in vivo without obvious side effects. Therefore, RgA is a novel GLUT-related chemical and promising target for preventing against HCC development and metastasis caused by diabetes-related risk factors.

Cancer cells often promote cancer development and progression by evading death receptor ligand-induced apoptosis[92]. Therefore, small molecules capable of restoring sensitivity to death receptor stimuli are an important tool for studying this biological pathway and may also provide evidence for developing potential drugs for cancer treatment. Using high-throughput screening, researchers have identified fasentin[93] with the function of selectively enhancing the sensitivity to fas cell surface death receptor and the tumour necrosis factor apoptosis-inducing ligand. Fasentin may interact with a unique site in the internal channel of GLUT1. This binding may interfere with the normal function of GLUT1, leading to partial inhibition of glucose transport. It alters the expressions of genes associated with nutrition and glucose deprivation, and inhibits glucose uptake, offering a novel mechanism for enhancing cellular sensitivity to death ligands. These findings may provide new strategies for cancer therapy, particularly in enhancing the effectiveness of death ligands such as tumor necrosis factor-α-related apoptosis-inducing ligands. Other inhibitors, such as WZB117 and BAY-876, may increase tumor sensitivity to chemotherapeutic agents by reducing glucose metabolism in tumour cells. They have shown potential inhibition of HCC cell growth and glucose uptake in vitro and in vivo[94]. Specifically, WZB117 has anti-tumour effects on various malignant cells as it can inhibit GLUT1 function to reduce intracellular glucose levels. WZB117 has been found to enhance the sensitivity of HCC cells to chemotherapeutic agents, especially in cisplatin-resistant cells. However, WZB117 is chemically unstable, leading to the emergence of a novel, more stable and effective second-generation anti-cancer agent, the pan-GLUT inhibitor DRB18[95]. Moreover, BAY-87681 can suppress HCC cell growth by inhibiting GLUT1-mediated glucose uptake. Recent research indicates that selective serotonin reuptake inhibitors inhibit HCC growth by targeting GLUT1, independently of their classical serotonin reuptake transporter target, and are associated with reversing the Warburg effect[96]. Isoginkgetin (ISO), a natural biflavonoid, induces autophagic cell death by targeting CDK6 and inhibiting SLC2A1/GLUT1 enhancer activity, thus inhibiting HCC cell proliferation and migration. This study offers a new molecular mechanism and theoretical basis for using ISO in HCC treatment[97]. Therefore, GLUT expression and activity may serve as biomarkers for diagnosing and prognosticating HCC, predicting treatment responses and disease progression. Developing inhibitors that target GLUTs, combined with other anticancer therapies such as chemotherapy, radiotherapy, and immunotherapy, can inhibit tumor growth, promote tumor cell apoptosis, and enhance tumor cell sensitivity to chemotherapy and radiotherapy by reducing glucose uptake in hepatocellular cancer cells (Table 1 and Figure 2).

Figure 2 Inhibitor action in the cell. Inhibitors reduce the uptake of glucose by tumour cells, reduce the ability of tumour cells to add value and induce apoptosis by competing with glucose transporter proteins (GLUTs) for glucose-binding targets and inhibiting the function of GLUTs. GLUT: Glucose transporter proteins.

Table 1 Currently common experimental drugs targeting glucose transporter proteins.

Ref.	Name	Target GLUTs	Ways to interfere with GLUT action
Ebstensen and Plagemann[86]	Cytochalasin B	Competition with GLUTs for binding sites	Reduces glucose supply to cancer cells
Ceballos et al[88]	Glupin	GLUT1, and GLUT3	Inhibition of glucose uptake
Karageorgis et al[89]	Chromopynones	GLUT1, and GLUT3	Inhibition of glucose uptake
Reckzeh et al[90]	Glutor	GLUT1, 2, and 3	Inhibits glycolytic processes
Guo et al[91]	Rapaglutin A	GLUT1, 3, and 4	Stimulates insulin secretion and inhibits GLUT uptake
Wood et al[93]	Fasentin	GLUT1	Inhibits glycolytic processes
Siebeneicher et al[94]	WZB117, BAY-876	GLUT1	Enhancing the sensitivity of HCC cells to chemotherapeutic agents

GLUT: Glucose transporter proteins; HCC: Hepatocellular carcinoma.

Anti-cancer strategies based on GLUTs in the Warburg effect include not only pharmacological compounds to block the action of GLUTs, but also dietary changes to mitigate the action glycolysis played in cancer cells. Ketogenic diet (KD) has been accepted to be a well-known adjuvant therapeutic strategy for cancer patients. KD refers to a high-fat, low-carbohydrate diet that contains sufficient protein to reduce the amount of glucose available to cancer cells throughout the body[98]. In addition to the traditional use in therapy-resistant epilepsy, KD can also benefit patients with glucose metabolism disorders such as glucose transporter 1 deficiency or pyruvate dehydrogenase deficiency[99]. Numerous clinical outcome studies have documented the effects of KD in slowing tumour growth, prolonging patient survival and reducing the resistance of cancer cells to conventional chemotherapy or radiotherapy[100]. It also protects healthy cells from chemotherapy/radiotherapy-induced damage and acts as an anti-inflammatory mechanism in the body[101]. However, in patients with HCC, especially those with fatty liver disease, KD should be used with caution and must be tailored to patients’ condition and used in conjunction with a GLUT inhibitor or other therapies to achieve optimal outcomes and improved prognosis.

Currently, several drugs and approaches are available to treat HCC by targeting and blocking GLUTs. Selective blockade of GLUTs in tumour cells remains a key challenge. GLUTs promote basal glucose uptake in most normal cell types and maintain glucose homeostasis under normal physiological conditions. In terms of the immune system, for example, immune cells also require glycolysis to maintain high proliferation rates for anti-cancer effects. The major obstacle in developing cancer cell metabolism-targeted drugs is to reduce toxicity and side effects on normal physiological cells. Blocking GLUTs alone can lead to GLUT deficiency and glucopenic brain injury[87]. GLUT1 deficiency syndrome is caused by impaired glucose transport across the blood-brain barrier into astrocytes, resulting in cerebral energy depletion. Co-administration of glucose disruptors with KD or dietary supplements (e.g., triheptanoin)[102] may be a solution to improve the safety of these compounds. For example, the results of a study[103] showed that using a novel active receptor-mediated complex, an adriamycin complex bound to a novel active receptor-mediated complex, ADM conjugated with 2-amino-2-deoxy-d-glucose and succinic acid (2DG-SUC-ADM) can target tumour cells via GLUT1, resulted in less toxicity to normal cells and might reverse drug resistance in tumour cells. 2DG-SUC-ADM could inhibit HCC HepG2 cells and stimulate apoptosis in vitro and in vivo. These known clinical trial data suggest that GLUTs inhibitors may be better used as adjuvant combination therapy for HCC. Anyway, more clinical trials of unearthing inhibitors targeting glucose transport should be initiated and expanded to discover adjuvant agents in cancer therapy[104].

CONCLUSION

This review indented to explore GLUTs in HCC and their role in tumour glucose metabolism, and to assess the possibility of GLUTs as a potential therapeutic target for HCC. GLUTs can facilitate glucose uptake and enhance glycolytic processes to support tumour growth and metabolic reprogramming in HCC cells. The expression of GLUTs is associated with inflammatory responses and immune escape in the TME. GLUT-targeted therapeutic strategies, such as GLUT inhibitors and RNA interference technologies targeting GLUTs, have shown anti-tumour activity both in vitro and in vivo. GLUTs are generally highly expressed in HCC, exhibiting possible relationships with tumour proliferation, invasion, and prognosis. GLUT1 and GLUT3 are two typical GLUTs in HCC, and their high expressions correlate with poor prognosis. This study also reviewed new developments in the study of GLUT2 in HCC. The expression of GLUTs in HCC is regulated by multiple factors, including epigenetic modifications, HIFs, and interactions with iron metabolism in the TME. Collectively GLUTs play key roles in glucose metabolism and tumour progression in HCC, and are potential targets for HCC treatment. Future studies need to further decipher the therapeutic pathways and potential of GLUTs in HCC.