Metabolic characteristics in hepatocellular carcinoma: amino acid metabolic reprogramming

Abstract

Hepatocellular carcinoma (HCC) is a common type of primary liver cancer and is considered the third leading cause of cancer-related deaths worldwide. The high aggressiveness and resistance to therapies exhibited by HCC present significant challenges to global public health. As the primary metabolic organ in the human body, the liver undergoes substantial metabolic reprogramming during carcinogenesis, affecting various metabolic pathways including those involved in carbohydrates, lipids, and amino acids. Notably, disruptions in amino acid metabolism play a critical role in the initiation and progression of HCC, helping to sustain its malignant characteristics. This review aims to provide an in-depth analysis of the alterations observed in aromatic amino acids metabolism, branched chain amino acids (BCAAs) metabolism, glutamine metabolism, and other amino acid metabolism processes, including serine, arginine, and methionine, along with the expression patterns of associated metabolic enzymes. Furthermore, it discusses potential therapeutic approaches and their clinical relevance, offering insights and strategies for improving HCC diagnosis and treatment in the future.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12943-025-02492-7.

Introduction

Hepatocellular carcinoma (HCC), a malignant tumor of the liver, is a primary cancer originating from hepatocytes and is one of the most common malignancies worldwide [1, 2]. According to the 2022 global cancer statistics, primary liver cancer is the sixth most frequently diagnosed cancer worldwide and the third leading cause of cancer-related mortality [3]. The elevated mortality rate of HCC can be attributed to difficulties in early detection, high metastasis rates in later stages, and the absence of safe and effective treatment options [4, 5]. Currently, the main risk factors for HCC include chronic infection with hepatitis B virus (HBV) and hepatitis C virus (HCV), aflatoxin exposure, alcohol abuse, and non-alcoholic fatty liver disease (NAFLD) related to diabetes or obesity [6–9]. However, with the widespread implementation of HBV vaccination and the use of antiviral therapies, the risk of HCC development related to viral liver diseases has significantly decreased [1]. In contrast, the rapid rise in NAFLD-related cases in recent years has made metabolic disorders an increasingly common risk factor for HCC development [10]. The reclassification of NAFLD to metabolic dysfunction-associated fatty liver disease (MAFLD) has attracted significant attention from biologists, pharmacologists, and clinical researchers [11]. The role of metabolic alterations in the progression of HCC is gaining increasing recognition, with non-viral factors such as obesity, diabetes, and metabolic syndrome likely becoming key areas of focus for future prevention, diagnosis, and treatment strategies [12]. Although significant advances in HCC treatment through extensive clinical exploration, HCC remains highly molecularly heterogeneous and complex due to its development involving somatic mutations, changes in the tumor microenvironment, and metabolic dysregulation [13]. Despite significant advances in curative treatments, HCC recurrence and metastasis rates remain high, leading to a poor overall prognosis. As a result, HCC presents a significant challenge for global healthcare systems in the development of new therapeutic approaches and strategies. A deeper understanding of HCC’s pathological mechanisms and effective disruption of its progression are crucial for improving patient outcomes [14, 15].

Cellular life processes are inextricably linked to metabolism, during which cells continuously consume a variety of specific nutrients, including carbohydrates, lipids, and amino acids. These substances act as the main sources of nutrition for maintaining energy balance and synthesizing macromolecules [16]. In contrast to normal cells, tumor cells undergo programmed alterations in their bioenergetics and metabolic pathways to fulfill the increased demands of rapid proliferation, biosynthesis, redox balance, and adaptation to environmental stress. This tumor-related programmatic metabolic shift is known as “metabolic reprogramming” [17]. Metabolic reprogramming is essential for tumor cell survival in hypoxic environments, as well as for various malignant transformations and subsequent invasion and migration [18, 19]. These metabolic changes involve numerous signaling pathways and alterations in the activity of key enzymes, primarily encompassing glycolysis, the pentose phosphate pathway, lipid metabolism, nucleic acid, and amino acid metabolism processes. The ultimate goals of these changes are to provide the energy and substrates necessary for tumor initiation and progression [20–22]. In recent years, accumulating evidence has highlighted the critical role of amino acid metabolic reprogramming in tumor progression [23]. Amino acids not only influence tumor cell proliferation, apoptosis, immune microenvironment, and chemoresistance, but also play pivotal roles at every stage of the tumor metastasis cascade [24, 25]. Moreover, amino acids mediate epigenetic regulation and post-transcriptional modifications within tumor cells [18, 26].

Under normal physiological conditions, the liver, being a central organ for protein synthesis and metabolism, contains a variety of non-essential amino acids, including glutamine, glutamate, and serine, alongside essential amino acids like leucine, phenylalanine, and tryptophan. These amino acids not only participate in hepatocyte metabolism but also contribute to lipid and nucleotide synthesis, as well as detoxification reactions [27, 28]. Additionally, they are crucial small molecules for protein synthesis in mammals and serve as precursors for certain bioactive compounds such as neurotransmitters and second messengers [29]. In the case of liver cancer, however, amino acids serve as an alternative energy source, fueling the rapid proliferation of tumor cells [30]. The development and progression of HCC require amino acids as nutrients, leading to an increased rate of amino acid metabolism, which generates sufficient bioactive substances, energy, and reducing agents to maintain redox balance and accommodate the abnormal proliferative demands of the tumor [23, 31]. Furthermore, amino acid metabolic reprogramming is closely associated with malignant characteristics of HCC, including metastasis, apoptosis, and chemoresistance. Therefore, this review will investigate the contribution of amino acid metabolism to the pathogenesis of HCC, aiming to elucidate the connection between amino acid metabolism and HCC progression. This understanding holds significant implications for clinical diagnosis, treatment, and prognosis of HCC in the future.

HCC and amino acid metabolism

Warburg and his team were the pioneers in identifying that tumor cells have a unique metabolic requirement for glucose as they progress. Specifically, most cancer cells depend on aerobic glycolysis to produce the necessary energy, a phenomenon referred to as the “Warburg effect.” [32]. Building on this discovery, researchers gradually revealed that the metabolic activities in tumor cells involve a series of micro-level changes, notably enhanced glycolysis, active lipid metabolism, and significantly altered uptake and utilization of certain amino acids (such as tyrosine and glutamine) compared to normal cells. Tumor cells utilize these metabolic changes to adapt to their abnormal proliferative characteristics [33]. For example, the transcription factor c-Myc enhances the activity of the pentose phosphate pathway by upregulating the expression of key enzymes such as glucose 6-phosphate dehydrogenase (G6PD), thereby meeting the biosynthetic needs of rapidly proliferating cancer cells and helping them cope with oxidative stress [34]. Yang et al. demonstrated that c-Myc stimulates cholesterol biosynthesis by increasing the transcription of SQLE, a key enzyme in the cholesterol synthesis pathway, which in turn boosts the proliferative potential of cancer cells [35]. Similarly, tumor cells exploit multiple metabolic pathways to strengthen their invasive and metastatic abilities. For instance, research has shown that lipid rafts, which are rich in cholesterol and sphingolipids in HCC, play a critical role in cell signaling [36]. These membrane microstructures promote HCC proliferation and migration by upregulating the expression of toll-like receptor 7 (TLR7) [37]. Aspartate synthetase (ASNS) facilitates the ATP-dependent conversion of aspartate and glutamine into asparagine and glutamate. In breast cancer cells, the increased activity of ASNS and its role in amino acid metabolism are strongly associated with cancer cell metastasis. Moreover, the levels of asparagine, a product of this metabolic pathway, can impact metastasis by modulating the epithelial-mesenchymal transition (EMT) process [38]. In addition, the metabolic process of nutrients also regulates tumor cell apoptosis, immune response in the microenvironment, and chemotherapy resistance through various pathways. Therefore, a deep understanding of the metabolic characteristics related to nutrients in tumor cells is essential for improving patient outcomes and increasing the effectiveness of treatments.

Carbohydrates, lipids, and amino acids, as the three primary energy sources, have metabolic processes closely linked to liver function. Abnormal liver function can lead to a series of metabolic imbalances, resulting in metabolic phenotypes distinct from those of normal cells, which influence the biological behavior of cells and ultimately cause further harm to the body. Amino acids, which are organic compounds containing both amine and carboxyl functional groups, serve as the fundamental units for protein synthesis in cells. As intermediate metabolites, they are involved in the biosynthesis of lipids, nucleotides, and polyamines [39]. The liver plays a crucial role in regulating amino acid metabolism. In individuals with HCC, these metabolic pathways are notably disrupted and are heavily implicated in various processes, including tumor proliferation, metastasis, apoptosis, and immune response modulation within the tumor microenvironment [40, 41]. Many studies have indicated that HCC is often accompanied by characteristic metabolic alterations in aromatic amino acids [42, 43], branched-chain amino acids [44], and glutamine [15], and maintaining homeostasis in these metabolic processes is crucial for HCC treatment, with the potential to serve as biomarkers and therapeutic targets [24]. Thus, understanding the link between amino acid metabolism and the onset and progression of HCC is essential for uncovering its pathogenesis and holds important implications for future clinical diagnosis and therapeutic strategies (Fig. 1).

Fig. 1.

Amino acids metabolism reprogramming in HCC

Aromatic amino acids metabolism

Aromatic amino acids (AAAs) are α-amino acids that contain a benzene ring in their molecular structure, which includes tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe). Phe and Tyr share similar structures, with Phe being converted into Tyr by phenylalanine hydroxylase (PAH) in the body. In recent years, AAAs have been extensively studied as key amino acids in metabolism. It has been found that patients with liver diseases (such as cirrhosis, fatty liver, and hepatitis) often exhibit elevated plasma levels of AAAs, particularly Phe and Tyr, compared to healthy individuals. More detailed and consistent metabolic signatures were observed in HCC patients. Specifically, in HCC patients, levels of Phe and Tyr in peripheral blood are elevated, while Trp levels are reduced [45–47]. Additionally, in the portal vein blood of HCC patients, the levels of Trp and Tyr notably elevated compared to healthy individuals, and the metabolism of Phe and Trp represents a major pathway of metabolite alterations in the portal vein [48]. Changes in the levels of AAAs in both serum and portal vein blood indicate abnormal metabolic activity in the liver tissue of HCC patients. Alterations in the metabolic pathways of Phe, Tyr, and Trp, as well as the levels of related metabolites, contribute to disease progression to a certain extent.

Tryptophan metabolism

Trp is an essential amino acid for humans and can only be obtained through daily dietary intake [49]. The levels of Trp in the body are influenced by dietary intake and the activity of its metabolic pathways. Beyond being a fundamental component for protein synthesis and a precursor for several important bioactive compounds, Trp plays vital roles in various physiological and pathological processes, including the regulation of cellular growth and proliferation [50], participating in immune and inflammatory responses [51], and regulating neuronal function [52, 53]. The active metabolites generated from Trp degradation are also widely involved in regulating the homeostasis of immune, neuronal, and gut functions, and these metabolites are considered potential therapeutic breakthroughs for cancer, neurodegenerative diseases, and other conditions [50, 53–55]. In recent years, increasing evidence has supported the link between Trp metabolic disorders and various diseases [56]. Trp and its related metabolites, along with key metabolic enzymes, influence various cellular biological functions through interactions with downstream molecules, affecting immune responses, cell proliferation, migration, and apoptosis [57]. Therefore, the characteristic alterations in Trp metabolism during diseases progression provide new insights for developing clinical therapeutic strategies.

Tryptophan metabolism process

Trp metabolism primarily occurs through three pathways: the indole pathway, the 5-hydroxytryptamine (5-HT) pathway, and the kynurenine (Kyn) pathway (Fig. 2) [58, 59]. In the indole pathway, a small portion of Trp is converted into indole compounds by tryptophanase in the gut microbiota, playing a role in maintaining gastrointestinal function, modulating inflammatory responses, antioxidation, and immune regulation [60]. Indole and its derivatives also play a role in regulating HCC, although current research in this area is still limited [61]. In the 5-HT pathway, a small fraction of Trp is hydroxylated by tryptophan hydroxylase (TPH) to produce 5-hydroxytryptophan (5-HTP), which is subsequently decarboxylated by aromatic L-amino acid decarboxylase (AADC) to form 5-HT, commonly referred to as serotonin. 5-HT is an important neurotransmitter that regulates vascular contraction and smooth muscle cell proliferation in peripheral tissues and influences mood, anxiety, and behavior in the central nervous system [62–64]. 5-HT is further transformed into melatonin through the action of hydroxyindole-O-methyltransferase (HIOMT) and serotonin-N-acetyltransferase (NAT), which exhibits antioxidant and antitumor properties and participates in regulating immune responses and circadian rhythms [65]. 5-HT is metabolized into 5-hydroxyindoleacetaldehyde (5-HIAL) through enzymatic reactions catalyzed by monoamine oxidase A (MAOA). Subsequently, 5-HIAL is converted into 5-hydroxyindoleacetic acid (5-HIAA) via the catalytic activity of aldehyde oxidase (AOX1) or aldehyde dehydrogenase 2 (ALDH2). This metabolite is associated with mood and behavior regulation and plays a role in the treatment of depression [66]. There is also a potential link between the 5-HT pathway and HCC progression. Different 5-HT receptors (5-HTRs) have varying roles in the development of HCC [67]. For example, 5-HT binding to 5-HTR1A inhibits hepatocyte DNA synthesis, thereby negatively regulating the growth of HCC cells [68]. In contrast, binding of 5-HT to 5-HTR1D activates the PI3K/Akt signaling pathway, which upregulates the expression of FoxO6, consequently enhancing the proliferation and migration of HCC cells both in vitro and in vivo [69].

Fig. 2.

The metabolic pathways of Trp in the human body include the kynurenine (Kyn), serotonin (5-HT), and indole pathways

The Kyn pathway is the most significant metabolic route for Trp in the human body, accounting for approximately 95% of its metabolism, and plays a crucial role in liver disease research [58, 70]. This pathway involves three key rate-limiting enzymes: tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase 1 (IDO1), and indoleamine 2,3-dioxygenase 2 (IDO2) (Fig. 2). Trp is initially converted into N-formylkynurenine (NFK) via three rate-limiting enzymes. NFK is subsequently transformed into Kyn through the action of arylformamidase. In the body, Kyn undergoes three major metabolic pathways: First, under the catalysis of kynurenine aminotransferase (KAT), Kyn is converted into kynurenic acid (KYNA). KYNA is a key non-competitive antagonist of the N-methyl-D-aspartate (NMDA) glutamate receptor, with potential endogenous antioxidant properties. It has the ability to inhibit excitotoxicity and neuroinflammation [71]. Although KYNA is typically considered neuroprotective, it has also been identified as an agonist for the aryl hydrocarbon receptor (AhR) and the orphan G protein-coupled receptor (GPR35), thereby influencing immune and inflammatory responses [72, 73]. Second, Kyn is converted into anthranilic acid (AA) through kynureninase (KYNU) activity. Third, Kyn is transformed into 3-hydroxykynurenine (3-HK) via kynurenine 3-monooxygenase (KMO). 3-HK is further metabolized into 3-hydroxyanthranilic acid (3-HAA) and alanine by KYNU, the latter of which is converted into pyruvate through transamination. Additionally, 3-HK can be converted into xanthurenic acid (XA) by KAT. Subsequently, 3-HAA is broken down into quinolinic acid (QuinA) via the catalytic activity of 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO), and QuinA is processed into the final product nicotinamide adenine dinucleotide (NAD+) through the action of quinolinic acid phosphoribosyltransferase (QPRT). This sequence of reactions constitutes the Kyn pathway, also known as the de novo NAD + synthesis pathway [74]. NAD + is a vital coenzyme in cellular energy metabolism, essential for controlling oxidative stress responses and supporting catabolic pathways. Moreover, NAD + impacts epigenetic processes by regulating the acetylation levels of histones and various other proteins [75].

Key enzymes in the kynurenine pathway

The key metabolic enzymes TDO, IDO1, and IDO2 exhibit a “double-edged sword” function in the body. On one hand, they suppress the growth of pathogens like Staphylococcus aureus and Toxoplasma gondii while regulating the body’s immune and inflammatory responses. On the other hand, they also facilitate immune suppression within the tumor microenvironment, aiding tumor immune escape and transplantation [76–79]. Among these enzymes, TDO, encoded by the TDO2 gene, is predominantly expressed in the liver, where it exerts a more significant catalytic effect under normal physiological conditions compared to IDO, responsible for degrading excess dietary Trp to maintain systemic Trp homeostasis [80]. In contrast, IDO1 and IDO2, encoded by the IDO1 and IDO2 genes respectively, are mainly distributed in extrahepatic tissues such as the brain, gastrointestinal tract, peripheral blood, and other immune cells. IDO1 and IDO2 have functional differences, with IDO1 being more efficient at metabolizing Trp than IDO2. While most current research focuses on IDO1, both IDO1 and IDO2 are collectively referred to as IDO here [81, 82]. IDO is activated by pro-inflammatory cytokines like interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), lipopolysaccharide (LPS), and others, playing a role in both innate and adaptive immune responses. In liver diseases, including bacterial and viral infections, autoimmune liver diseases, liver tumors, and liver transplantation, these pro-inflammatory cytokines strongly induce IDO expression. Due to its roles in both infection control and tumor promotion, IDO has garnered considerable attention, explaining its broader relevance in liver diseases compared to TDO [83–85]. In fact, numerous studies have confirmed that dysregulation of the Kyn pathway is common across various cancers, particularly colorectal cancer, pancreatic cancer, and gliomas. Similar issues have been observed in HCC [69, 86–89]. The upregulation of key rate-limiting enzymes TDO and IDO in HCC leads to Trp depletion and increased Kyn levels in the serum and tumor microenvironment. Alterations in the Kyn/Trp ratio are closely linked to the onset and progression of diseases and have been recognized as a factor contributing to poor prognosis in patients [90–93].

Research has shown that TDO was initially discovered in human glioma cells and has since been detected in various human tumors, including HCC, bladder cancer, and breast cancer [94]. The overexpression of TDO is positively associated with the malignancy of HCC [95]. In mouse tumor models, TDO overexpression prevents immune rejection, while systemic treatment of mouse with TDO small-molecule inhibitors restores immune rejection [96]. The relapse-free survival rate in IDO-negative HCC patients is considerably lower compared to IDO-positive patients [97]. Uyttenhove et al. also demonstrated in the P815 mouse tumor model that high IDO expression effectively counteracts immune rejection [98]. At the microscopic level, the hyperactivity of TDO and IDO in HCC catalyzes the tryptophan-kynurenine metabolic pathway, leading to Trp depletion in the tumor microenvironment. This promotes “metabolic immune regulation,” ultimately resulting in strong suppression of the immune surveillance system, allowing HCC cells to evade immune detection and survive [51]. CD69, an immune regulatory molecule, is primarily expressed by early activated leukocytes at chronic inflammation sites. CD69 + T cells promote IDO expression in monocytes and tumor-associated macrophages by releasing IFN-γ, thereby inducing immune tolerance in HCC cells by counteracting T-cell responses in the tumor microenvironment [99]. Additionally, TDO and IDO promote the recruitment of regulatory T cells into the tumor microenvironment, thereby enhancing tumor immune tolerance and facilitating HCC metastasis [100, 101]. However, some studies have reported that TDO expression in HCC is lower than in adjacent normal tissues [91, 102]. Cheng et al. discovered that the loss of TDO is associated with shortened survival in HCC patients. They also found that TDO inhibits cell cycle progression by increasing the expression of cell cycle arrest proteins P21 and P27, leading to G1 phase arrest in HCC cells and thereby suppressing their proliferation [95]. The reasons for these discrepant findings are likely multifactorial. First, TDO shows tissue-specific expression: unlike its low basal levels and aberrant induction in other cancers, it is constitutively expressed in the liver to sustain tryptophan homeostasis. Thus, its downregulation in HCC may indicate loss of hepatocyte differentiation, resembling tumor suppressor loss rather than oncogenic activation. Second, methodological differences—detection techniques, antibody specificity, and cut-off definitions for expression levels—may contribute to discrepancies. Finally, patient and tumor heterogeneity, including diverse etiologies, stage, grade, and liver function, may differentially affect TDO patterns. The lack of clear distinction between tumor, immune, and stromal sources of TDO further complicates interpretation. Therefore, the exact role of TDO in HCC remains controversial.

Metabolites in the kynurenine pathway

The bioactive metabolites generated in the Kyn pathway, such as Kyn, KYNA, 3-HK, 3-HAA, and QuinA, also contribute to tumor cell proliferation, tissue invasion and metastasis, as well as the regulation of local immune function within the tumor microenvironment (Fig. 3) [55, 91, 103, 104]. Kyn, as an endogenous ligand promoting tumor proliferation, can specifically activate the aryl hydrocarbon receptor (AhR) via autocrine or paracrine signaling. AhR, a ligand-activated transcription factor belonging to the basic helix-loop-helix (bHLH)/PAS protein family, is expressed in many immune cells and can be activated by indoles, indole derivatives, and Kyn. Activated AhR regulates various immune functions within cells and is associated with tumorigenesis and invasion, while blocking AhR signaling can improve patient prognosis [61, 105]. Numerous studies have highlighted the critical role of the Kyn-AhR signaling axis in HCC progression. This pathway suppresses immune cell activity, leading to impaired immune responses against tumors and the induction of immune tolerance [105, 106]. Specifically, AhR promotes the differentiation of regulatory T cells (Tregs), thereby inhibiting anti-tumor immune responses [66]. Opitz et al. found that in TDO and AhR high-expressing mouse models, glioma cells enhance their growth by inhibiting antitumor immune cell infiltration and increasing inflammatory cytokine levels, while this effect was not observed in AhR-deficient mice [91]. Liu et al. studied the role of zinc finger transcription factor ZNF165 from the Kruppel family in Trp signaling and metabolism, revealing that ZNF165 promotes HCC cell proliferation and migration by regulating the Trp/Kyn/AhR/CYP1A1 axis [107]. Additionally, Kyn can directly activate AhR through CYP1A1 or CYP1B1 [66]. Notably, Kyn generated by IDO can activate AhR and stimulate IL-6 production, which subsequently promotes IDO expression via STAT3 activation. This suggests that human cancer cells can maintain their IDO expression via an autocrine IDO-AhR-IL-6-STAT3 loop, thereby promoting HCC growth and survival [108]. Moreover, AhR also promotes HCC cell proliferation and EMT through the activation of the STAT3 and NF-κB/TIM4 pathways [101].

Fig. 3.

Simplified overview of Trp metabolism in HCC

Apart from Kyn, KYNA is also an endogenous ligand for AhR. Studies have shown that KYNA significantly enhances the colony formation, migration, and invasion abilities of MHCC-97 H, Huh7, and PLC/PRF/5 cell lines under heat stress by activating AhR [109]. Interestingly, KYNA can be produced not only through Kyn metabolism but also via interleukin-4-induced-1 (IL4L1), further promoting cancer cell motility and suppressing adaptive immunity [110]. Other downstream metabolites, including 3-HK, 3-HAA, and QuinA, have also been demonstrated to inhibit T cell proliferation and activation, thereby enhancing the immune evasion capacity of tumor cells. 3-HK and 3-HAA, as part of the Kyn pathway, exhibit clear immunosuppressive properties. Specifically, 3-HK inhibits the maturation and proliferation of CD8 + T cells, while 3-HAA suppresses T cell proliferation by interfering with the activation of critical signaling pathways, such as NF-κB [111, 112]. Moreover, 3-HAA negatively regulates T cell activation by inhibiting T cell receptor (TCR)-mediated calcium signaling and promotes the formation of regulatory T cells (Tregs) through the stimulation of transforming growth factor-β (TGF-β) [113]. Notably, 3-HAA also regulates apoptosis in HCC cells via the activation of the transcription factor YY1, thereby influencing the fate of tumor cells [114]. QuinA, although primarily known for its neurotoxic effects, also plays a significant role in immune regulation. It selectively induces apoptosis in Th1-type helper T cells and influences the balance between Th17 cells and Tregs, thereby promoting immune suppression within the tumor microenvironment [115]. Collectively, these metabolites create an immunosuppressive microenvironment by inhibiting T cell activation and proliferation, enhancing the tumor’s ability to evade the immune system. This immune suppression occurs not only through direct actions on T cells but also by indirectly affecting dendritic cells and other immune system components [79]. These mechanisms are critical for effective immune surveillance and the immune rejection of tumors.

IDO expression is induced by cytokines such as INF-γ, contributing to immune tolerance within the tumor microenvironment. TDO inhibits cell proliferation by upregulating cell cycle arrest proteins P21 and P27, thus halting cell cycle progression. Tryptophan metabolites, such as Kyn, modulate various immune functions in HCC cells via the Kyn/AhR signaling pathway and are associated with tumor proliferation, invasion, and immune tolerance. The metabolite 3-HAA suppresses T-cell proliferation through inhibition of key pathways, such as NF-κB, exerting an immunosuppressive effect. Additionally, 3-HAA promotes the generation of regulatory T cells by stimulating the production of TGF-β and regulates apoptosis in HCC cells via activation of the transcription factor YY1. Indole and indole derivatives also activate AhR, thereby regulating various immune functions within the cells. Furthermore, 5-HT promotes the proliferation and migration of HCC cells by binding to 5-HTR1D and activating the PI3K/Akt/FoxO6 pathway. This schematic provides a simplified overview of the key pathways discussed, and is not intended to be an exhaustive representation of all metabolic interactions.

Phenylalanine/tyrosine metabolism

Tyrosine metabolism

Tyr is a conditionally essential ketogenic and glucogenic amino acid, whose catabolism primarily occurs in the liver, with a portion also taking place in the kidneys [116]. As an intermediate or precursor in the tricarboxylic acid (TCA) cycle and ketogenic process, Tyr and its metabolites play essential roles in human growth, development, and metabolism [72, 117]. The degradation of Tyr relies on a series of enzyme-catalyzed reactions. In the normal human liver, five highly expressed catabolic enzymes include tyrosine aminotransferase (TAT), 4-hydroxyphenylpyruvate dioxygenase (HPPD), homogentisate 1,2-dioxygenase (HGD), glutathione S-transferase zeta 1 (GSTZ1), and fumarylacetoacetate hydrolase (FAH) [117–119]. Deficiency in these enzymes leads to the accumulation of toxic tyrosine metabolites, such as 4-hydroxyphenylpyruvate, homogentisic acid, and fumarylacetoacetate, in the blood, liver cells, or urine, which reduces glutathione levels, causes DNA and cellular damage, and triggers apoptosis. Tyr has been investigated as a potential cancer biomarker and is strongly correlated with the progression of various cancer types [120, 121]. In pathological conditions, alterations in tyrosine metabolism typically involve abnormal expression of some catabolic tyrosine enzymes in the human body. For example, decreased fumarylacetoacetate hydrolase (FAH) expression affects the sensitivity of epithelial ovarian cancer (EOC) to DNA-damaging agents, reducing the cytotoxicity of these agents against EOC and undermining the efficacy of genotoxic chemotherapy in treating EOC [122]. Additionally, the downregulation of HGD and GSTZ1 is linked to enhanced aerobic glycolysis in kidney renal clear cell carcinoma (KIRC), which subsequently alters the balance of amino acid and energy metabolism in tumor cells. This disruption ultimately activates the cell cycle and accelerates tumor progression [123]. In advanced-stage HCC patients, the expression of all five tyrosine catabolic enzymes is reduced, leading to elevated serum Tyr levels. The diminished expression of these enzymes is strongly correlated with poorer survival outcomes in HCC patients [124, 125]. Therefore, investigating the regulatory mechanisms of tyrosine catabolic enzymes in HCC has become a focus of research for scientists.

Enzymes in tyrosine metabolism

TAT is the first rate-limiting enzyme in the tyrosine metabolic pathway, responsible for converting Tyr to 4-hydroxyphenylpyruvate (4-HPP). Dysfunction of TAT can lead to type II tyrosinemia. Research has shown that loss of the TAT gene and inactivation due to hypermethylation are closely associated with the development of HCC. Functional studies have demonstrated that, as a mitochondrial protein, TAT exerts strong tumor-suppressive effects by promoting the release of cytochrome C, activating caspase-9 and poly (ADP-ribose) polymerase (PARP), and thereby inducing apoptosis through mitochondrial pathways [125]. Furthermore, the mechanism through which TAT induces apoptosis in HCC cells may be associated with TGF-β signaling. TAT activates the TGF-β pathway, triggering the phosphorylation of Smad2 and Smad3. The phosphorylated Smad2/3 then forms a complex with Smad4, which translocates to the nucleus to regulate TAT gene expression, thereby further activating caspase-9 and promoting apoptosis in HCC cells [126]. HPPD is a Fe (II)-dependent non-heme dioxygenase that plays a key role in catalyzing the conversion of 4-HPP to homogentisate (HGA) in the liver [127]. Deficiency in HPPD causes Type III tyrosinemia. In HCC, HPPD acts as a tumor suppressor gene, and its expression is inhibited. Studies have shown that decreased HPPD expression under disease conditions is linked to poor overall survival, highlighting its potential clinical relevance as a prognostic marker in HCC patients. Yang et al. further confirmed at the cellular level that HPPD exerts its tumor-suppressive effects by inhibiting the proliferation and migration of HCC cells [128]. However, the precise contribution of HPPD in carcinogenesis and tumor metabolism remains unclear, with most HPPD-related research focusing on microbial metabolism and herbicide applications, leaving limited studies on its role in human tumors. Glutathione S-transferases (GSTs) are a class of phase II metabolic isozymes that protect cells from toxic molecules by conjugating them with glutathione. Specific GSTs also participate in the regulation of stress signals related to cell proliferation and apoptosis [129]. GSTZ1, a member of the glutathione S-transferase (GST) family, belongs to the zeta 1 class and is also known as maleylacetoacetate isomerase (MAA1). It catalyzes the GSH-dependent isomerization of maleylacetoacetate (MAA) into fumarylacetoacetate (FAA) [130]. GSTZ1 is the fourth enzyme in tyrosine catabolism and one of the most extensively studied in this pathway. As a key tumor suppressor, GSTZ1 is markedly downregulated in HCC and is strongly correlated with poor prognosis in patients [131, 132]. HCC is characterized by enhanced aerobic glycolysis and limited oxidative phosphorylation activity, contributing to reduced apoptosis and cancer progression. Studies have shown that increased GSTZ1 expression downregulates glycolysis-related genes, promotes oxidative phosphorylation and electron transport, and thus suppresses HCC progression [124]. GSTZ1 is also implicated in oxidative stress [133]. GSTZ1 deficiency results in GSH depletion, leading to increased reactive oxygen species (ROS) levels and enhanced lipid peroxidation. This triggers the activation of the NRF2/KEAP1 (nuclear factor erythroid 2–related factor 2/Kelch-like ECH-associated protein 1)-mediated antioxidant pathway. NRF2 binds to antioxidant response elements (ARE), leading to the upregulation of downstream target genes such as NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1), which in turn promotes HCC cell proliferation and aerobic glycolysis [132]. NRF2 is a key regulator of cellular detoxification and antioxidant responses, playing a role in tumor initiation, metastasis, and chemoresistance [134]. Several studies have highlighted the persistent activation of NRF2 in various cancers, including liver, lung, ovarian, and gastric cancers [135]. The activation of the NRF2 pathway due to GSTZ1 deficiency can affect HCC through multiple mechanisms. For instance, GSTZ1 deficiency activates the NRF2 pathway, increasing the expression of ferroptosis-related genes such as glutathione peroxidase 4 (GPX4) and cystine/glutamate transporter (SLC7A11/xCT), thereby inhibiting sorafenib-induced ferroptosis and contributing to chemoresistance in HCC cells. The combination of sorafenib and the GPX4 inhibitor RSL3 has been shown to significantly suppress the viability of GSTZ1-deficient cells and induce ferroptosis in HCC cells [136].

In addition, the deficiency of GSTZ1 leads to the accumulation of succinylacetone (SA), activating the NRF2/IGF1R axis. This mechanism involves the alkylation of KEAP1 and subsequent activation of NRF2, which interacts with the zinc finger transcription factor SP1 to promote its enrichment on the insulin-like growth factor 1 receptor (IGF1R) promoter. This increases IGF1R transcription, mediates the PI3K/Akt and Ras/ERK signaling pathways, and enhances the proliferation, differentiation, and anti-apoptotic abilities of HCC cells both in vitro and in vivo, ultimately promoting HCC progression [131]. The alkylation of KEAP1 and activation of NRF2 due to GSTZ1 deficiency also promote HCC cell migration through the glucuronic acid pathway. This pathway, primarily located in the cytosol of liver cells, serves as an alternative oxidative route for glucose metabolism. In this process, UDP-glucose dehydrogenase (UGDH) converts UDP-glucose (UDP-Glc) into UDP-glucuronic acid (UDP-GlcUA), and the accumulation of its metabolic products is linked to increased cell adhesion and migration. GSTZ1 deficiency leads to UDP-GlcUA accumulation, stabilizing TGFβR1 mRNA by enhancing the interaction between TGFβR1 and PTBP3, which in turn activates TGFβ/Smad signaling and promotes HCC migration [137]. The gene SLC27A5, encoding fatty acid transport protein 5 (FATP5), is highly expressed in the liver and responsible for transporting extracellular fatty acids into hepatocytes for lipid synthesis. It is also an upstream regulator of the expression of several tyrosine metabolic enzyme genes. In HCC, the expression of SLC27A5 is significantly suppressed, leading to decreased hepatic fatty acid uptake. This suppression affects the nuclear translocation of the transcription factor NRF2, inhibiting the transcription of tyrosine metabolic enzymes and thereby influencing the tyrosine metabolism process, promoting HCC cell cycle progression [118]. FAH catalyzes the breakdown of FAA into fumarate (FUMA) and acetoacetate (ACA), the final step in tyrosine degradation. Type I tyrosinemia (HT1) is caused by congenital FAH deficiency, characterized by liver dysfunction, cirrhosis, and an increased risk of HCC [138]. FUMA and ACA are converted into acetyl-CoA through glucose and lipid metabolism, respectively [139]. Acetyl-CoA is a crucial intermediate in energy metabolism, playing a central role in the TCA cycle and oxidative phosphorylation, while also serving as a precursor for the synthesis of fatty acids, ketone bodies, and cholesterol (Fig. 4). Therefore, abnormalities in tyrosine metabolism can alter the flux of other metabolic pathways, eventually leading to disease [118]. In HCC, FAH mRNA expression is significantly reduced, potentially contributing to disease progression [124]. However, the exact mechanisms remain largely unexplored and require further investigation.

Fig. 4.

Phenylalanine/tyrosine metabolism in HCC

Phenylalanine metabolism

Phenylalanine (Phe) is an essential amino acid for humans, and its metabolism is primarily catalyzed by PAH, converting Phe into tyrosine (Fig. 4) [140]. The resulting tyrosine further participates in the synthesis of various bioactive substances such as neurotransmitters and hormones. Additionally, Phe can be metabolized into phenylacetic acid and phenylactic acid, among other metabolites. Abnormal Phe metabolism is associated with several inherited metabolic disorders, including phenylketonuria (PKU), tyrosinemia, and albinism [141, 142]. Studies have reported that the tumor-associated inflammation and immune activation commonly observed in cancer patients can disrupt Phe metabolism by inhibiting PAH activity, leading to elevated Phe levels [143]. In HCC, Phe metabolism is similarly affected. Compared with normal liver tissue, PAH expression is significantly reduced in HCC patients, leading to increase Phe levels in peripheral blood. High PAH expression is correlated with favorable clinical outcomes in HCC patients, suggesting that PAH could serve as a prognostic biomarker. The E3 ubiquitin ligase APC/Cdh1 interacts with PAH via the 26 S proteasome pathway, promoting PAH polyubiquitination and thus downregulating its function and stability. In HCC tissues, the expression of PAH is significantly downregulated compared to normal tissues, whereas Cdh1 expression is markedly upregulated, suggesting a potential clinical correlation between Cdh1 and PAH. Based on these findings, Cdh1 and PAH may be potential therapeutic targets for improving HCC prognosis [144, 145].

The expression of six key metabolic enzymes—phenylalanine hydroxylase (PAH), tyrosine aminotransferase (TAT), 4-hydroxyphenylpyruvate dioxygenase (HPPD), homogentisate 1,2-dioxygenase (HGD), glutathione S-transferase zeta 1 (GSTZ1), and fumarylacetoacetate hydrolase (FAH)—is downregulated. The E3 ubiquitin ligase APC/Cdh1 interacts with PAH via the 26 s proteasome pathway, promoting PAH degradation and contributing to HCC progression. Transforming growth factor-beta (TGF-β) signaling triggers the phosphorylation of Smad2/3, resulting in the nuclear translocation of Smad2/3 and Smad4, which regulate TAT gene expression. This signaling cascade facilitates the release of cytochrome C from mitochondria, activates caspase-9, and initiates apoptosis in HCC cells. The loss of GSTZ1 function activates the NRF2/KEAP1-mediated antioxidant pathway. NRF2 binds to antioxidant response elements (ARE), enhancing the expression of target genes such as NQO1, which promotes HCC cell proliferation. NRF2 also interacts with the zinc finger transcription factor SP1, regulating IGF1R transcription and mediating the PI3K/Akt and Ras/ERK signaling pathways, thus enhancing HCC cell proliferation and anti-apoptotic capacities. Moreover, NRF2 activation promotes HCC cell migration through the glucuronidation pathway and induces chemoresistance by upregulating the expression of glutathione peroxidase 4 (GPX4).

Drugs and clinical trials

In HCC and associated immune cells, the high expression of IDO and TDO promotes the activation of the tryptophan metabolic pathway, affecting HCC proliferation, migration, and the regulation of its immune microenvironment. Developing inhibitors of the Kyn pathway could help improve disease progression and prognosis in HCC patients [146]. Given the dysregulated tryptophan metabolism in disease, inhibitors targeting the tryptophan metabolic pathway have been under investigation. Several drugs targeting key metabolic enzymes such as TDO and IDO have been tested in clinical trials for cancer, with some having completed trials (Table 1, Source from website: https://clinicaltrials.gov/). Unfortunately, studies have shown limitations in using IDO or TDO inhibitors alone as therapeutic agents, while combination therapies with other clinical drugs have shown better efficacy. For example, the IDO inhibitor indoximod (1-Methyl-D-tryptophan, D-1MT) can act as a Trp mimetic and has demonstrated short-term tumor growth delay in preclinical and clinical trial data, but its use as a standalone therapy remains controversial [147–150]. However, combining D-1MT with various chemotherapeutic agents (cisplatin, cyclophosphamide, doxorubicin, etc.) has effectively promoted the regression of chemotherapy-resistant cancers, such as breast cancer [90]. Additionally, combining the IDO/TDO inhibitor IACS-8968 with temozolomide (TMZ) enhances TMZ’s cytotoxicity in glioma cells, improving the efficacy of chemotherapy [151]. The IDO inhibitor Abrine, when combined with anti-PD-1 antibodies, inhibits tumor growth by upregulating CD4 + and CD8 + T cells, downregulating Foxp3 + Treg cells, and inhibiting both IDO and PD-L1 expression [152]. A TDO-targeting conjugate composed of the TDO inhibitor PVI and the camptothecin derivative irinotecan exerts dual effects by inducing apoptosis and promoting T-cell proliferation, showcasing the synergy between immunotherapy and chemotherapy [153]. Epacadostat, one of the most studied IDO inhibitors, exerts anticancer effects by competing with tryptophan, a Kyn pathway substrate, for binding to the enzyme’s catalytic domain. It exhibits potent anti-IDO activity by enhancing the activation of T cells and natural killer (NK) cells, while suppressing Treg function [154]. Epacadostat also inhibits Treg proliferation induced by IFN-γ and lipopolysaccharide (LPS)-treated human dendritic cells. T-cells activated by dendritic cells treated with Epacadostat show a marked increase in IFN-γ production and demonstrate enhanced effectiveness in in vitro tumor cell lysis assays [155]. However, in vivo studies have shown that Epacadostat monotherapy does not produce significant anticancer activity, but its combination with immune checkpoint inhibitors has yielded promising results. Combination therapy appears to have good tolerability. Initial phase I/II clinical data indicate that the combination of Epacadostat and Pembrolizumab (an anti-PD-1 antibody) enhances overall response rates in patients with non-small cell lung cancer (NSCLC), bladder cancer, and squamous cell carcinoma of the head and neck (SCCHN) [156–158]. This success has paved the way for Epacadostat’s advancement into phase III clinical trials.

Table 1.

 Clinical Study on Trp, Gln, and Arg Metabolism in Cancer Treatment

Target	NCT Number	Study Title	Interventions	Phases
IDO	NCT05843448	IDO and PD-L1 Peptide Based Immune-Modulatory Therapeutic (IO102-IO103) in Combination With Pembrolizumab for BCG-Unresponsive or Intolerant, Non-Muscle Invasive Bladder Cancer	BIOLOGICAL: PD-L1/IDO Peptide Vaccine|BIOLOGICAL: Pembrolizumab	PHASE1
	NCT03915405	KHK2455 (IDO Inhibitor) Plus Avelumab in Adult Subjects With Advanced Bladder Cancer	DRUG: KHK2455|DRUG: Avelumab	PHASE1
	NCT05077709	IO102-IO103 in Combination With Pembrolizumab as First-line Treatment for Patients With Metastatic NSCLC, SCCHN, or mUBC	DRUG: IO102-IO103 in combination with pembrolizumab	PHASE2
	NCT03343613	A Study of LY3381916 Alone or in Combination With LY3300054 in Participants With Solid Tumors	DRUG: LY3381916|DRUG: LY3300054	PHASE1
	NCT02460367	Immunotherapy Combination Study in Advanced Previously Treated Non-Small Cell Lung Cancer	DRUG: Docetaxel|BIOLOGICAL: Tergenpumatucel-L|DRUG: Indoximod	PHASE1
	NCT01685255	A Phase 2 Study of the IDO Inhibitor Epacadostat Versus Tamoxifen for Subjects With Biochemical-recurrent-only EOC, PPC or FTC Following Complete Remission With First-line Chemotherapy	DRUG: Epacadostat|DRUG: tamoxifen	PHASE2
	NCT02118285	Intraperitoneal Natural Killer Cells and INCB024360 for Recurrent Ovarian, Fallopian Tube, and Primary Peritoneal Cancer	DRUG: Fludarabine|DRUG: Cyclophosphamide|BIOLOGICAL: NK cells|BIOLOGICAL: IL-2|DRUG: INCB024360	PHASE1
	NCT04190498	Sleep Apnoea Syndrome and Hepatocellular Carcinoma	DIAGNOSTIC_TEST: Nocturnal oximetry
	NCT03695250	BMS-986205 and Nivolumab as First or Second Line Therapy in Treating Patients With Liver Cancer	DRUG: IDO1 Inhibitor BMS-986205|BIOLOGICAL: Nivolumab	PHASE1|PHASE2
	NCT03361865	Pembrolizumab in Combination With Epacadostat or Placebo in Cisplatin-ineligible Urothelial Carcinoma (KEYNOTE-672/ECHO-307)	DRUG: Pembrolizumab|DRUG: Epacadostat|DRUG: Placebo	PHASE3
	NCT02077881	Study of IDO Inhibitor in Combination With Gemcitabine and Nab-Paclitaxel in Patients With Metastatic Pancreatic Cancer	DRUG: Nab-Paclitaxel|DRUG: Gemcitabine|DRUG: Indoximod	PHASE1|PHASE2
	NCT03374488	Pembrolizumab + Epacadostat vs Pembrolizumab + Placebo in Recurrent or Progressive Metastatic Urothelial Carcinoma	DRUG: Pembrolizumab|DRUG: Epacadostat|DRUG: Placebo	PHASE3
	NCT03348904	Nivolumab and Epacadostat With Platinum Doublet Chemotherapy Versus Platinum Doublet Chemotherapy in Non-Small Cell Lung Cancer	DRUG: Nivolumab|DRUG: Epacadostat|DRUG: Placebo|DRUG: Carboplatin|DRUG: Cisplatin|DRUG: Gemcitabine|DRUG: Paclitaxel|DRUG: Pemetrexed	PHASE3
	NCT03347123	A Study of Epacadostat and Nivolumab in Combination With Immune Therapies in Participants With Advanced or Metastatic Malignancies (ECHO-208)	DRUG: Epacadostat|DRUG: Nivolumab|DRUG: Ipilimumab|DRUG: Lirilumab	PHASE1|PHASE2
	NCT03342352	Nivolumab Plus Epacadostat in Combination With Chemotherapy Versus the EXTREME Regimen in Squamous Cell Carcinoma of the Head and Neck (CheckMate 9NA/ECHO-310)	DRUG: Nivolumab|DRUG: Epacadostat|DRUG: Placebo|DRUG: Carboplatin|DRUG: Cisplatin|DRUG: Cetuximab|DRUG: 5-Fluorouracil	PHASE3
	NCT03854032	Nivolumab and BMS986205 in Treating Patients With Stage II-IV Squamous Cell Cancer of the Head and Neck	BIOLOGICAL: Nivolumab|BIOLOGICAL: IDO1 Inhibitor BMS-986205|PROCEDURE: Therapeutic Conventional Surgery|OTHER: Questionnaire Administration	PHASE2
	NCT03896113	Neoadjuvant Celecoxib in Newly Diagnosed Patients With Endometrial Carcinoma	DRUG: Celecoxib 200mg capsule	PHASE2
	NCT02166905	DEC-205/NY-ESO-1 Fusion Protein CDX-1401, Poly ICLC, and IDO1 Inhibitor INCB024360 in Treating Patients With Ovarian, Fallopian Tube, or Primary Peritoneal Cancer in Remission	BIOLOGICAL: DEC-205/NY-ESO-1 Fusion Protein CDX-1401|DRUG: Epacadostat|OTHER: Laboratory Biomarker Analysis|OTHER: Pharmacological Study|DRUG: Poly ICLC	PHASE1|PHASE2
	NCT01982487	Vaccine Therapy and IDO1 Inhibitor INCB024360 in Treating Patients With Epithelial Ovarian, Fallopian Tube, or Primary Peritoneal Cancer Who Are in Remission	BIOLOGICAL: ALVAC(2)-NY-ESO-1 (M)/TRICOM vaccine|DRUG: IDO1 inhibitor INCB024360|OTHER: laboratory biomarker analysis|OTHER: pharmacological study	PHASE1|PHASE2
	NCT05280314	Phase II Trial of Neoadjuvant and Adjuvant IO102-IO103 and Pembrolizumab KEYTRUDAin Patients With Resectable Tumors	DRUG: IO102-IO103|DRUG: Pembrolizumab KEYTRUDA	PHASE2
	NCT02042430	Epacadostat Before Surgery in Treating Patients With Newly Diagnosed Stage III-IV Epithelial Ovarian, Fallopian Tube, or Primary Peritoneal Cancer	DRUG: Epacadostat|OTHER: Laboratory Biomarker Analysis|PROCEDURE: Therapeutic Conventional Surgery	EARLY_PHASE1
	NCT03260894	Pembrolizumab (MK-3475) Plus Epacadostat vs Standard of Care in mRCC (KEYNOTE-679/ECHO-302)	DRUG: Pembrolizumab|DRUG: Epacadostat|DRUG: Sunitinib|DRUG: Pazopanib	PHASE3
	NCT03322540	Pembrolizumab Plus Epacadostat vs Pembrolizumab Plus Placebo in Metastatic Non-Small Cell Lung Cancer (KEYNOTE-654-05/ECHO-305-05)	DRUG: Pembrolizumab|DRUG: Epacadostat|DRUG: Placebo	PHASE2
	NCT03358472	Pembrolizumab Plus Epacadostat, Pembrolizumab Monotherapy, and the EXTREME Regimen in Recurrent or Metastatic Head and Neck Squamous Cell Carcinoma (KEYNOTE-669/ECHO-304)	DRUG: Pembrolizumab|DRUG: Epacadostat|DRUG: Cetuximab|DRUG: Cisplatin|DRUG: Carboplatin|DRUG: 5-Fluorouracil	PHASE3
	NCT03322566	A Study of Pembrolizumab Plus Epacadostat With Platinum-based Chemotherapy Versus Pembrolizumab Plus Platinum-based Chemotherapy Plus Placebo in Metastatic Non-Small Cell Lung Cancer (KEYNOTE-715-06/ECHO-306-06)	DRUG: Pembrolizumab|DRUG: Epacadostat|DRUG: Platinum-based chemotherapy|DRUG: Placebo	PHASE2
	NCT03325465	Neoadjuvant Pembrolizumab + Epacadostat Prior to Curative Surgical Care for Squamous Cell Carcinoma of the Head and Neck	DRUG: Pembrolizumab|DRUG: Epacadostat	PHASE2
	NCT04463771	Safety and Efficacy of Retifanlimab (INCMGA00012) Alone or in Combination With Other Therapies in Participants With Advanced or Metastatic Endometrial Cancer Who Have Progressed on or After Platinum-based Chemotherapy.	DRUG: retifanlimab|DRUG: epacadostat|DRUG: pemigatinib|DRUG: INCAGN02385|DRUG: INCAGN02390	PHASE2
GLS	NCT02071862	Study of the Glutaminase Inhibitor CB-839 in Solid Tumors	DRUG: CB-839|DRUG: Pac-CB|DRUG: CBE|DRUG: CB-Erl|DRUG: CBD|DRUG: CB-Cabo	PHASE1
	NCT05521997	Glutaminase Inhibition and Chemoradiation in Advanced Cervical Cancer	DRUG: Telaglenastat|RADIATION: Radiation treatment|DRUG: Cisplatin	PHASE2
	NCT03944902	CB-839 in Combination With Niraparib in Platinum Resistant BRCA -Wild-type Ovarian Cancer Patients	DRUG: Cohort 1: Dose Escalation|DRUG: Cohort 2: Dose Escalation	PHASE1
	NCT03163667	CB-839 With Everolimus vs. Placebo With Everolimus in Participants With Renal Cell Carcinoma (RCC)	DRUG: Placebo|DRUG: CB-839|DRUG: everolimus	PHASE2
	NCT03428217	CANTATA: CB-839 With Cabozantinib vs. Cabozantinib With Placebo in Patients With Metastatic Renal Cell Carcinoma	DRUG: CB-839|DRUG: Cabozantinib|DRUG: Placebo	PHASE2
	NCT02771626	Study CB-839 in Combination With Nivolumab in Patients With Melanoma, Clear Cell Renal Cell Carcinoma (ccRCC) and Non-Small Cell Lung Cancer (NSCLC)	DRUG: CB-839|DRUG: Nivolumab	PHASE1|PHASE2
	NCT03875313	Study of CB-839 (Telaglenastat) in Combination With Talazoparib in Patients With Solid Tumors	DRUG: CB-839|DRUG: Talazoparib	PHASE1|PHASE2
	NCT04250545	Testing of the Anti Cancer Drugs CB-839 HCl (Telaglenastat) and MLN0128 (Sapanisertib) in Advanced Stage Non-small Cell Lung Cancer	DRUG: Sapanisertib|DRUG: Telaglenastat Hydrochloride	PHASE1
	NCT05688215	Zimberelimab and Quemliclustat in Combination with Chemotherapy for the Treatment of Patients with Borderline Resectable and Locally Advanced Pancreatic Adenocarcinoma	PROCEDURE: Biospecimen Collection|PROCEDURE: Computed Tomography|PROCEDURE: Core Biopsy|DRUG: Fluorouracil|DRUG: Irinotecan|DRUG: Leucovorin|DRUG: Leucovorin Calcium|DRUG: Oxaliplatin|DRUG: Quemliclustat|DRUG: Zimberelimab	PHASE1|PHASE2
	NCT04265534	KEAPSAKE: A Study of Telaglenastat (CB-839) With Standard-of-Care Chemoimmunotherapy in 1L KEAP1/NRF2-Mutated, Nonsquamous NSCLC	DRUG: Telaglenastat|DRUG: Carboplatin Chemotherapy|DRUG: Pemetrexed Chemotherapy|BIOLOGICAL: Pembrolizumab Immunotherapy|DRUG: Placebo|DIETARY_SUPPLEMENT: Folic acid 400 -1000 g|DIETARY_SUPPLEMENT: Vitamin B12 1000 g|DRUG: Dexamethasone 4 mg	PHASE2
	NCT03831932	Telaglenastat Hydrochloride and Osimertinib in Treating Patients With EGFR-Mutated Stage IV Non-small Cell Lung Cancer	PROCEDURE: Biospecimen Collection|PROCEDURE: Computed Tomography|PROCEDURE: Magnetic Resonance Elastography|DRUG: Osimertinib|PROCEDURE: Positron Emission Tomography|DRUG: Telaglenastat Hydrochloride|PROCEDURE: X-Ray Imaging	PHASE1|PHASE2
	NCT04698681	NGS Screening Protocol to Detect Mutation of KEAP1 or NRF2/NFE2L2 Genes for the KEAPSAKE (CX-839-014) Trial
Arg	NCT02006030	Ph 2 Trial of ADI PEG 20 Plus Concurrent Transarterial Chemoembolization (TACE) Vs TACE Alone in Patients With Unresectable Hepatocellular Carcinoma	DRUG: ADI-PEG 20|DRUG: Transarterial chemoembolization	PHASE2
	NCT03278444	Treatment of Advanced Hepatocellular Carcinoma	OTHER: Basic drugs therapy of HCC|DRUG: Arginine hydrochloride|DRUG: Trimetazidine hydrochloride	PHASE3
	NCT02029690	Ph 1 Study in Subjects With Tumors Requiring Arginine to Assess ADI-PEG 20 With Pemetrexed and Cisplatin	DRUG: ADI-PEG 20	PHASE1
	NCT02102022	Ph 1-2 Study ADI-PEG 20 Plus FOLFOX in Subjects With Advanced GI Malignancies Focusing on Hepatocellular Carcinoma	DRUG: ADI-PEG 20 plus modified FOLFOX6	PHASE1|PHASE2
	NCT02101593	Ph 1 Trial of ADI PEG 20 Plus Sorafenib to Treat Patients With Liver Cancer	DRUG: ADI-PEG 20	PHASE1
	NCT06034977	Phase 2 Study of ADI-PEG 20 Plus Lenvatinib Treatment in Subjects With Unresectable Hepatocellular Carcinoma	DRUG: ADI-PEG20	PHASE2
	NCT03236935	Phase Ib of L-NMMA and Pembrolizumab	DRUG: L-NMMA|DRUG: Pembrolizumab	PHASE1
	NCT03274427	Treatment of Intermediate-stage Hepatocellular Carcinoma	DEVICE: Basic drugs therapy of HCC by TACE|DRUG: Arginine hydrochloride|DRUG: Trimetazidine hydrochloride	PHASE3
	NCT03498222	Study in Patients With Tumours Requiring Arginine to Assess ADI-PEG 20 With Atezolizumab, Pemetrexed and Carboplatin	DRUG: Atezolizumab|DRUG: Pemetrexed|DRUG: Carboplatin|DRUG: ADI PEG20	PHASE1
	NCT01665183	Ph 1 Trial of ADI-PEG 20 Plus Cisplatin in Patients With Metastatic Melanoma	DRUG: ADI-PEG 20	PHASE1
	NCT05616624	ADI-PEG 20 in Combination With Gemcitabine and Docetaxel After Progression on Frontline Therapy in Non-small Cell and Small Cell Lung Cancers	DRUG: ADI-PEG 20|DRUG: Gemcitabine|DRUG: Docetaxel	PHASE1|PHASE2
	NCT03950518	Novel Treatment of Advanced Hepatocellular Carcinoma	DRUG: Anlotinib Hydrochloride Capsules|DRUG: Arginine hydrochloride|DRUG: levamisole	PHASE3
	NCT01287585	Ph 3 ADI-PEG 20 Versus Placebo in Subjects With Advanced Hepatocellular Carcinoma Who Have Failed Prior Systemic Therapy	DRUG: ADI-PEG 20 (arginine deiminase formulated with polyethylene glycol)|DRUG: Placebo|OTHER: Best Supportive Care	PHASE3
	NCT04965714	Nivolumab and ADI-PEG 20 Before Surgery for the Treatment of Resectable Liver Cancer	BIOLOGICAL: Nivolumab|BIOLOGICAL: Pegargiminase|PROCEDURE: Resection	PHASE2
	NCT01266018	Study of ADI-PEG 20 in Patients With Relapsed Sensitive or Refractory Small Cell Lung Cancer	DRUG: ADI-PEG 20 (Arginine deiminase pegylated)	PHASE2
	NCT05317819	Study of ADI-PEG 20 Versus Placebo in Subjects With High Arginine Level and Unresectable Hepatocellular Carcinoma	DRUG: ADI-PEG20|OTHER: Placebo	PHASE3
	NCT02285101	Recombinant Human Arginase 1 (rhArg1) in Patients With Advanced Arginine Auxotrophic Solid Tumors	BIOLOGICAL: PEG-BCT-100	PHASE1
	NCT01314755	A Trial of Perioperative Immune Enhancing Feed in Patients Undergoing Surgery for Head and Neck Cancer	DIETARY_SUPPLEMENT: IMPACT|DIETARY_SUPPLEMENT: An iso-caloric, iso-nitrogenous control feed	NA
	NCT03449901	ADI-PEG 20 in Combination With Gemcitabine and Docetaxel for the Treatment of Soft Tissue Sarcoma, Osteosarcoma, Ewing's Sarcoma, and Small Cell Lung Cancer	DRUG: pegylated arginine deiminase|DRUG: Gemcitabine|DRUG: Docetaxel|PROCEDURE: Tumor biopsy|PROCEDURE: Research blood draw	PHASE2
	NCT00559156	Arginine/Omega-3 Fatty Acids/Nucleotides Nutritional Supplement in Treating Patients With Stage III or Stage IV Head and Neck Cancer Undergoing Chemotherapy and Radiation Therapy	DIETARY_SUPPLEMENT: arginine/omega-3 fatty acids/nucleotides oral supplement|DRUG: cisplatin|PROCEDURE: adjuvant therapy|RADIATION: radiation therapy	PHASE2
	NCT00151671	Effect of a Perioperative Oral Nutritional Supplementation on Patients Undergoing Hepatic Surgery for Liver Cancer	DRUG: Oral Impact |DRUG: Placebo	PHASE2|PHASE3
	NCT04001543	Efficacy of an Oral Immunomodulatory Nutrient on Survival During Postoperative Concomitant Chemoradiotherapy in Head and Neck Cancer	DIETARY_SUPPLEMENT: Immunomodulating oral supplementation|DIETARY_SUPPLEMENT: Sip feed control	NA
	NCT01806675	18F-FPPRGD2 PET/CT or PET/MRI in Predicting Early Response in Patients With Cancer Receiving Anti-Angiogenesis Therapy	DRUG: 18F-fludeoxyglucose (18F-FDG)|DRUG: 18F-FPPRGD2	PHASE1|PHASE2
	NCT01256034	Effects of Preoperative Immunonutrition in Patients Undergoing Pancreaticoduodenectomy	DIETARY_SUPPLEMENT: Oral IMPACT	PHASE4
	NCT05498428	A Study of Amivantamab in Participants With Advanced or Metastatic Solid Tumors Including Epidermal Growth Factor Receptor (EGFR)-Mutated Non-Small Cell Lung Cancer	DRUG: Amivantamab|DRUG: Lazertinib|DRUG: Carboplatin|DRUG: Pemetrexed|DRUG: Direct Oral Anticoagulant (DOAC)|DRUG: Low Molecular Weight Heparin (LMWH)	PHASE2
	NCT01256047	Effects of Preoperative Immunonutrition in Patients Undergoing Hepatectomy	DIETARY_SUPPLEMENT: Oral IMPACT	PHASE4
	NCT02277197	Ficlatuzumab and Cetuximab in Recurrent/Metastatic Head and Neck Squamous Cell Carcinoma (HNSCC)	DRUG: Ficlatuzumab|DRUG: Cetuximab	PHASE1
	NCT02277184	Ficlatuzumab, Cisplatin and IMRT in Locally Advanced Head and Neck Squamous Cell Carcinoma	DRUG: Ficlatuzumab|DRUG: Cisplatin|RADIATION: Intensity Modulated Radiotherapy (IMRT)	PHASE1

In conclusion, the overexpression of IDO and TDO in Trp metabolism is closely linked to immune suppression, proliferation, and invasion in HCC, suggesting that targeting the Trp metabolic pathway could be a novel and promising adjunctive strategy for treating HCC. Preclinical data indicate that pharmacological inhibition of TDO and IDO may represent a safe and effective cancer treatment. By enhancing the immune system’s ability to destroy tumors, these inhibitors can improve the efficacy of cancer immunotherapy. TDO/IDO inhibitors have been tested in clinical trials, both as standalone treatments and in combination with chemotherapy or monoclonal antibodies, with results demonstrating that combination therapy is an effective strategy. Thus, targeting the Trp metabolic pathway in HCC holds great promise as an adjunctive therapy, and further exploration of its mechanisms is needed to facilitate its broader clinical application. On the other hand, the dysregulation of Phe/Tyr metabolism has been shown to be associated with various chronic diseases and cancers [159]. In HCC, reduced expression of key enzymes involved in the Phe/Tyr metabolic pathway is associated with poor patient survival, with GSTZ1 playing a particularly prominent role. Nevertheless, the mechanisms of Phe/Tyr metabolic reprogramming and its clinical application in HCC require further investigation to provide a solid theoretical foundation for future therapeutic strategies.

Branched chain amino acids (BCAAs) metabolism

BCAAs consist of three amino acids with branched side chains: leucine (Leu), isoleucine (Ile), and valine (Val). While BCAAs can be synthesized by bacteria, plants, and fungi, higher animals, including humans, cannot synthesize these amino acids. Nonetheless, BCAAs are prevalent in mammals, making up about 35% of the essential amino acids in muscle protein [160]. In humans, BCAAs serve as substrates for synthesizing nitrogen-containing compounds and are involved in various physiological processes, including protein synthesis and metabolism, energy homeostasis, gut function regulation, and immune responses [161, 162].

Metabolic process of BCAAs

BCAAs metabolism mainly occurs in muscles and the liver, with the liver playing a critical role in BCAAs oxidation and protein synthesis [163]. Since the liver has low activity of the first enzyme in the BCAAs degradation pathway, branched-chain amino acid aminotransferases (BCATs), the initial steps of BCAAs catabolism primarily take place in extrahepatic tissues, particularly in muscle [162]. In extrahepatic tissues, BCAAs are transported into the cytosol via L-type amino acid transporters (LATs) and then undergo reversible transamination catalyzed by branched-chain amino acid transferase 1 (BCAT1) in the cytosol or BCAT2 in mitochondria. This reaction produces corresponding branched-chain α-keto acids (BCKAs) and glutamate (Glu) [28, 163, 164]. Glu can act as an amino donor and participate in reactions with pyruvate (Pyr) to form alanine (Ala) and α-ketoglutarate (α-KG) or serve as a detoxifying agent by generating glutamine (Gln), with most of the Ala and Gln being released into the bloodstream. The resulting BCKAs, including α-ketoisocaproic acid (KIC), α-ketoisovaleric acid (KIV), and α-ketomethylvaleric acid (KMV), can be used for energy supply or transported to the liver for further oxidation and metabolism [165–167]. The liver plays a pivotal role in the subsequent reactions of BCAAs metabolism, where the branched-chain α-keto acid dehydrogenase (BCKDH) complex, located on the inner mitochondrial membrane, catalyzes the irreversible oxidative decarboxylation of BCKAs from muscle and other tissues. This process is essential for maintaining blood glucose levels and overall energy balance [168]. BCKDH activity is tightly regulated by branched-chain α-keto acid dehydrogenase kinase (BCKDK) and Mg2+/Mn2+-dependent protein phosphatase 1 K (PPM1K) [169]. BCKDK inhibits BCKDH by phosphorylating BCKDHA, a subunit of BCKDH, while PPM1K dephosphorylates BCKDHA, activating BCKDH and thus regulating BCAAs metabolism [170]. Ultimately, the metabolism of KIV produces succinyl-CoA, while KIC is metabolized into acetyl-CoA and acetoacetate, or it can be converted into 3-hydroxyisobutyric acid (HMB) via the enzyme KIC dioxygenase. KMV is metabolized to generate both acetyl-CoA and succinyl-CoA. The end products, acetyl-CoA and succinyl-CoA, enter the TCA cycle to produce ATP, whereas HMB is converted into cholesterol (Fig. 5) [171–173].

Fig. 5.

Metabolic process of BCAAs in vivo

The role and treatment of BCAAs in HCC

Although the biological effects of these three amino acids differ, their primary metabolic pathways are shared, and most research examines BCAAs mixtures. In 1971, Fisher et al. first introduced the concept of the Fisher ratio, which represents the molar ratio of BCAAs to AAAs in plasma [174]. Multiple studies have shown that this ratio is crucial for evaluating liver metabolism, liver function, and the extent of liver damage. In patients with liver disease, particularly liver cirrhosis, the Fisher ratio typically decreases and is associated with the occurrence of hepatic encephalopathy (HE) [175]. Several metabolomics studies have also confirmed a correlation between circulating BCAAs levels and various liver diseases [176, 177]. As the second most abundant nitrogen source in cells, following glutamine, BCAAs metabolism dysfunction likely represents a “crossroad” in disease development. Studies have indicated that impaired BCAAs metabolism is associated with the development of diseases such as diabetes, cardiovascular disease, and cancer [168, 178]. In HCC patients, circulating BCAAs levels are elevated and have been identified as biomarkers for HCC progression [179, 180]. Additionally, BCAAs levels in HCC tissue are significantly higher than in non-tumor tissues. These findings suggest that BCAAs metabolism may be a key factor in the development of HCC.

BCAAs function as important signaling molecules in multiple signaling networks, particularly the PI3K/Akt/mTOR pathway, which regulates glucose, lipid, and protein synthesis, maintains gut health, and modulates immune responses. In recent years, increasing evidence has shown that BCAAs are involved in PI3K/Akt/mTOR signaling in various tissues, including the liver. For example, in HCC cell models cultured under long-term high insulin conditions, BCAAs supplementation can inhibit the PI3K/Akt signaling pathway by inducing a negative feedback loop through mTORC1/S6K1 activation. Additionally, BCAAs can enhance apoptosis in HCC cells by inhibiting mTORC2 kinase activity on Akt, thereby influencing HCC progression [181]. It is well-known that mTOR is a central regulator of cellular responses to nutrients and growth factors, and it signaling pathway is frequently overactivated in many cancers, including HCC [182, 183]. mTOR exists in two distinct complexes: mTORC1 and mTORC2, each with different structures and functions. mTORC1 regulates protein translation and enhances the translation mechanism through two primary effectors: ribosomal protein S6 kinase (p70S6K, S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) [184, 185]. On the other hand, mTORC2 phosphorylates Akt at Ser473 and is insensitive to rapamycin. Studies suggest that dual inhibition of mTORC1 and mTORC2 has a more significant impact on tumor growth [186]. For example, leucine (Leu) activates mTORC1 by binding to the sensor Sestrin2. This activation triggers a cascade of downstream signaling pathways, including the phosphorylation of effectors such as 4EBP1 and S6K1, which regulate protein and lipid synthesis. Through this process, leucine promotes cellular protein synthesis and inhibits autophagy [187, 188]. Due to the activation of mTORC1 by BCAAs, they are sometimes thought to potentially promote tumor progression in liver disease patients. However, in the context of insulin-induced overactivation of the PI3K/Akt signaling pathway, studies have shown that BCAAs can inhibit tumor progression through the aforementioned mechanisms. Furthermore, BCAAs can suppress apoptosis signaling through the Wnt/β-catenin pathway and fibrotic signaling through the NFY/TGF-β1/Smad2/3 pathway, which are induced by histone acetyltransferase P300 in hepatic stellate cells (HSCs), reducing the risk of HCC [189]. HSCs are known to contribute to liver fibrosis, and their persistent activation results in the secretion of TGF-β1 and collagen, leading to liver cirrhosis and ultimately HCC. Overexpression of the β-catenin signaling pathway also promotes the expression of oncogenes such as cyclin D1 and c-Myc [190]. These mechanisms are strongly linked to the progression of various cancers, including HCC. In cardiomyocytes, BCAAs metabolites like BCKA can also enhance apoptotic susceptibility by downregulating mTORC2 downstream effectors, including Akt and protein kinase C (PKC) [191]. This highlights the diverse effects of BCAAs on different tissues and their involvement in crucial signaling pathways.

However, the involvement of BCAAs metabolism in the onset and progression of HCC remains a topic of debate. Previous studies have indicated that BCAAs may have both tumor-promoting and tumor-suppressing effects on cancer. On the one hand, some studies have shown that BCAAs supplementation can reduce the incidence of liver cancer in obese cirrhotic patients or in cancer models induced by obesity and diabetes [192]. Additionally, oral BCAAs supplementation significantly lowered the risk of HCC and reduced liver-related events in patients with cirrhosis [193]. In vitro, studies have demonstrated that BCAAs inhibit insulin-mediated HepG2 cell proliferation by reducing the expression of insulin-like growth factor-1 receptor (IGF-1R) [161]. On the other hand, an increasing body of evidence suggests that BCAAs might also promote cancer development through certain mechanisms [194–196]. For instance, Ericksen et al., through transcriptomic and metabolomic analyses, highlighted that the gradual loss of BCAAs catabolism is closely related to HCC growth and progression. In HCC, the inhibition of BCAAs catabolic enzyme expression leads to BCAAs accumulation in tumors, and this metabolic process is involved in regulating mTORC1 activity, affecting HCC growth [44]. Yang et al. found that under Gln deprivation conditions, HCC cells activate the BCAAs catabolism pathway to promote cell cycle progression and survival. Mechanistically, increased O-GlcNAcylation under Gln deprivation stabilizes the PPM1K protein, which dephosphorylates BCKDHA, enhances BCAAs degradation and ultimately promoting HCC progression, which is associated with poor patient prognosis [197]. Changes in BCAAs metabolism not only affect the state of cancer cells but also have profound impacts on the systemic metabolism of individuals with malignant tumors [170]. These contradictory findings may be due to specific environmental conditions, such as the tissue origin and nutritional status, both of which can influence the consequences of BCAAs metabolism in the body [196, 198]. The varying activity of BCAAs metabolism under different conditions suggests that HCC cells may employ compensatory mechanisms during BCAAs metabolism. This highlights the complexity of BCAAs metabolism in HCC and the importance of considering the broader metabolic context when evaluating its role in tumor development and treatment.

Today, BCAAs have been extensively studied as a clinical supplement, playing a significant role in promoting protein synthesis, inhibiting protein breakdown, and enhancing glutamine synthesis [199]. A randomized, double-blind clinical trial evaluating the efficacy of BCAAs supplementation during radiotherapy showed that oral BCAAs improved biochemical markers and amino acid profiles in HCC patients, helping to alleviate malnutrition and improve patient prognosis [200]. Additionally, dietary intake of BCAAs has been shown to prevent the recurrence of HCC after radiofrequency ablation and supportive treatments, while also protecting liver function [201]. For cirrhotic patients, oral BCAAs supplementation not only improves nutritional status and reduces the risk of developing HCC but also enhances the activity of neutrophils and natural killer (NK) cells, effectively reducing the risk of bacterial and viral infections in patients with decompensated cirrhosis [202]. A meta-analysis of patients undergoing HCC interventions revealed that those who received BCAAs supplementation had better liver function reserves, higher serum albumin levels, lower rates of ascites and edema, and an overall improved quality of life compared to patients who did not receive BCAAs [203]. These findings suggest that BCAAs supplementation can offer a range of clinical benefits for HCC and cirrhosis patients, making it a valuable component in managing liver-related conditions.

Enzymes in BCAAs metabolism

Additionally, BCAT1, a key enzyme in the initial stage of BCAAs catabolism, has been demonstrated to play a significant role in the development and progression of various cancers. BCAT1 regulates tumor cell behavior through signaling pathways such as PI3K/Akt/mTOR, RAS/ERK, and Wnt/β-catenin in different tumor types [204]. For example, BCAT1 provides metabolic plasticity to triple-negative breast cancer cells, allowing them to modify their reliance on the RAS/ERK and PI3K/Akt/mTOR signaling cascades in response to IGF-1/insulin, thereby promoting tumor survival and progression [205]. In HCC patients, high expression of BCAT1 is associated with poor prognosis, indicating its potential as a molecular target for the diagnosis and treatment of HCC [206, 207]. The oncogenic role of BCAT1 in HCC involves the stimulation of cellular proliferation, migratory capacity, and metastatic potential through the activation of the Akt signaling cascade and induction of EMT. Conversely, BCAT1 knockdown upregulates E-cadherin expression and inhibits the expression of Twist and vimentin, thus suppressing the EMT process in HCC cells [208]. Moreover, cisplatin-induced BCAT1 upregulation reduces HCC cell sensitivity to cisplatin by modulating mTOR-mediated autophagy in BCAAs metabolism, contributing to chemotherapy resistance [209]. c-Myc, a well-known oncogene and transcription factor, is widely involved in the development and progression of various cancers. Research findings indicate a significant association between elevated BCAT1 levels and increased c-Myc expression. Knocking down c-Myc in HCC cells leads to downregulation of BCAT1, and reducing BCAT1 expression significantly inhibits the migration and invasion of HCC cells [206]. In addition, BCKDK, which regulates the activity of BCKDH, mediates HCC cell proliferation and migration through the APN/BCKDK/ERK axis. Aminopeptidase A (APN) is upregulated in HCC tissues and highly metastatic cell lines, and its gene knockout suppresses HCC cell proliferation and metastasis. Functional studies have shown that APN deficiency activates the ERK signaling pathway. APN mediates the phosphorylation of BCKDK at serine, promoting the interaction between BCKDK and ERK1/2, thus activating the ERK signaling pathway in HCC cells. Therefore, the APN/BCKDK/ERK axis may represent a novel therapeutic target for HCC [210]. (Fig. 6)

Fig. 6.

BCAAs metabolism in HCC

In summary, the role of BCAAs metabolism in the development of HCC has become a focal point of research in recent years. Currently, the most clearly defined mechanism linking BCAAs to cancer involves the activation of the mTORC1 signaling pathway. Additionally, the specific expression of enzymes within this pathway may serve as potential targets for intervention in cancer therapy. However, the exact relationship between BCAAs metabolism and HCC remains complex, and the effects of BCAAs supplementation in HCC are still not conclusively determined, with further research needed to explore underlying mechanisms. Although the use of BCAAs-related biomarkers in cancer screening and diagnosis is still in the early stages of exploration, future research could focus on the potential of BCAAs as biomarkers combined with targeted cancer therapies. In summary, the role of BCAAs metabolism in the development of HCC has become a focal point of research in recent years. Currently, the most clearly defined mechanism linking BCAAs to cancer involves the activation of the mTORC1 signaling pathway. Additionally, the specific expression of enzymes within this pathway may serve as potential targets for intervention in cancer therapy. However, the exact relationship between BCAAs metabolism and HCC remains complex, and the effects of BCAAs supplementation in HCC are still not conclusively determined, with further research needed to explore underlying mechanisms. Although the use of BCAAs-related biomarkers in cancer screening and diagnosis is still in the early stages of exploration, future research could focus on the potential of BCAAs as biomarkers combined with targeted cancer therapies. It is important to note that while the three amino acids within BCAAs share similar metabolic pathways and functions, it is not yet clear in most diseases whether their roles are dependent on the effects of individual BCAAs, and this deeper mechanism warrants further investigation in future studies. This could help clarify the specific contributions of each BCAAs and potentially lead to more targeted therapeutic approaches based on their distinct biological impacts.

BCAAs play a role in regulating the PI3K/Akt/mTOR pathway. BCAAs inhibit PI3K/Akt signaling via a negative feedback loop induced by mTORC1/S6K1 activation, while also suppressing Wnt/β-catenin apoptotic signaling mediated by histone acetyltransferase P300 and the NFγ/TGF-β1/Smad2/3 fibrosis signaling pathway through mTORC1 activation. Leucine (Leu) activates mTORC1 by binding to Sestrin2, leading to the phosphorylation of downstream effectors 4EBP1 and S6K1, promoting protein synthesis and inhibiting autophagy. Knocking out the BCAT1 gene upregulates E-cadherin expression in HCC cells and inhibits the expression of Twist and Vimentin, thereby suppressing cell migration. Branched-chain keto acid dehydrogenase kinase (BCKDK) inhibits BCKDH activity through phosphorylation, while PPMIK activates BCKDH via dephosphorylation. Additionally, BCKDK promotes HCC cell proliferation and migration through the APN/BCKDK/ERK axis.

Glutamine metabolism

Metabolic process of glutamine

Gln, as the amide form of glutamate, is one of the most abundant amino acids in the bloodstream and serves as an important carbon source for anabolic processes and energy production. The primary source of Gln for the human body is dietary intake, with up to 30% of dietary Gln retained by intestinal epithelial cells. Although glutamine synthetase enables muscles and other organs to synthesize Gln de novo, it is still classified as a non-essential amino acid under normal physiological conditions. However, under conditions of catabolic stress—such as post-surgery, trauma, or sepsis—Gln becomes a conditionally essential amino acid, with tissues like the kidneys and gastrointestinal tract, especially intestinal mucosal cells, significantly increasing Gln consumption. Depletion of Gln in these cells leads to rapid necrosis [211, 212]. In cells, Gln enters through the solute carrier family 1 member 5 (SLC1A5, also known as ASCT2) and is converted to glutamate by glutaminase (GLS) or glutaminase 2 (GLS2) in the mitochondria. Glutamate plays a crucial role in the synthesis of glutathione (GSH), which helps maintain cellular redox homeostasis [213, 214]. Subsequently, glutamate is further converted into α-KG through the catalytic actions of glutamate dehydrogenase (GDH), alanine transaminase (ALT), or aspartate transaminase (AST). These catalytic processes also produce other amino acids and ammonium ions [215]. α-KG enters the TCA cycle, generating ATP to fuel cellular functions [15, 22]. Beyond energy production, Gln metabolism supports various biosynthetic pathways. Oxaloacetate from the TCA cycle can be converted into aspartate to facilitate nucleotide synthesis, malate is catalyzed by malic enzyme 1 (ME1) to produce NADPH and pyruvate, with NADPH serving as reducing equivalents [216, 217]. Additionally, α-KG can be converted into citrate through the reverse reductive carboxylation catalyzed by isocitrate dehydrogenase 2 (IDH2), producing acetyl-CoA, which is exported to the cytosol for lipid synthesis (Fig. 6) [218].

Given that Gln metabolism is involved in multiple biosynthetic pathways, cancer cells often enhance Gln metabolism to meet the demands of DNA, protein, and lipid synthesis for rapid proliferation [219]. Recent studies have revealed that HCC is particularly dependent on Gln metabolism. Gln serves as a critical nitrogen source for amino acid and nucleotide synthesis in HCC cells. Additionally, a significant portion of Gln is converted into GSH, which acts as the primary antioxidant in HCC cells. Through Gln metabolism, the increased GSH levels reduce reactive oxygen species (ROS), enhancing the cell’s antioxidant defenses and protecting against oxidative stress [220]. Therefore, targeting Gln metabolism holds great therapeutic potential for HCC. The pathways involved in Gln metabolism offer important targets for developing more effective HCC treatment strategies.

Enzymes in glutamine metabolism

GLS is a key enzyme in glutamine catabolism and exists in mammalian cells in two isoforms: GLS1 (or GLS) and GLS2. These isoforms show distinct expression patterns in different tissues. The GLS1 gene encodes two isoforms, kidney-type glutaminase (KGA) and glutaminase C (GAC). GLS1 is closely associated with tumor growth and proliferation, and inhibiting GLS1 activity has been shown to slow tumor growth, disrupt redox homeostasis in cancer cells, and thereby inhibit tumor progression. In contrast, GLS2, encoded by the GLS2 gene, functions as a tumor suppressor in certain contexts, with its isoforms glutaminase B (GAB) and liver-type glutaminase (LGA) playing key roles [221–224]. In normal liver cells, GLS2 is preferentially expressed, but in HCC, there may be a MYC-dependent metabolic shift from GLS2 to GLS1, facilitating glutamine metabolism rewiring to support tumor cell proliferation [15, 225]. In fact, many studies have shown that GLS1 plays a crucial role in cancer progression, participating in the occurrence and development of various malignant phenotypes. GLS1 can enhance tumor proliferation, invasion, and migration by maintaining redox homeostasis, energy metabolism, and proliferation signaling pathways. Silencing GLS1 can significantly inhibit the proliferation and invasion of many cancers, including pancreatic cancer, osteosarcoma and HCC [221, 226–229]. In HCC, GLS1 is highly expressed compared to healthy tissues and is involved in the regulation of various mechanisms. For example, research indicates that GLS1 promotes HCC cell proliferation through activation of the Akt/GSK3β/CyclinD1 pathway [230]. Moreover, GLS1 is a key substrate in the hepatocyte growth factor (HGF)/MET receptor tyrosine kinase signaling pathway. The HGF/MET axis stimulates glycolysis (Warburg effect) and glutamine breakdown in HCC cells by activating GLS1, which maintains proliferation, promotes invasion and metastasis, and contributes to chemotherapy resistance [231]. SMYD2, a methyltransferase associated with various cancers, is upregulated in HCC tissues and is linked to poor clinical outcomes. c-Myc, a well-known oncogenic transcription factor, stimulates glutamine metabolism by upregulating GLS1, supporting cancer cell growth [232]. Studies have shown that SMYD2 can reprogram glutamine metabolism via the c-Myc/GLS1 axis to promote HCC progression. Mechanistically, SMYD2 methylates c-Myc, increasing its stability via the ubiquitin-proteasome system in HCC, leading to GLS1 upregulation and enhanced glutamine metabolism, which promotes proliferation and chemoresistance [233]. This reveals the therapeutic potential of targeting SMYD2 and GLS1 in HCC patients. Cancer stem cells (CSCs), which drive tumor recurrence, metastasis, and chemoresistance, are also closely associated with GLS1 expression. GLS1 is upregulated in liver CSCs, promoting glutamate production, which serves as a precursor for GSH, regulating intracellular ROS levels and oxidative stress responses. By consuming ROS, GSH reduces ROS levels, enhancing β-catenin protein translocation and upregulating stemness-related genes in HCC, which correlates with poor prognosis. Targeting GLS1 has been shown to diminish HCC stemness by increasing ROS and inhibiting the Wnt/β-catenin pathway [234]. Lee et al. demonstrated that HepG2 cells with cancer stem cell characteristics exhibit a lower dependency on glucose and primarily rely on mitochondrial oxidative phosphorylation driven by glutamine metabolism to meet their energy demands. This glutamine dependency supports the high ATP requirements needed for P-glycoprotein (P-gp)-mediated drug efflux. Combining doxorubicin, the mitochondrial inhibitor metformin, and glutamine deprivation has been shown to enhance chemosensitivity in HepG2 cells [235]. In contrast to the oncogenic role of GLS1, GLS2 expression is negatively correlated with advanced HCC, tumor recurrence, overall survival, and disease-free survival. Mechanistic studies have revealed that GLS2 stabilizes Dicer through the ubiquitination system, promoting the maturation of miR-34a. Mature miR-34a inhibits Snail expression, reducing HCC cell invasion and the EMT process [236]. These findings underscore the dual roles of GLS1 and GLS2 in HCC progression and highlight their potential as therapeutic targets in HCC treatment.

GDH is also a key mitochondrial enzyme in glutamine metabolism, primarily found in the liver, myocardium, and kidneys, with smaller amounts in the brain, skeletal muscle, and leukocytes. Human GDH exists in two isoforms: hGDH1, which is encoded by the GLUD1 gene located on chromosome 10 and is expressed across all tissues, with the highest levels found in the liver; and hGDH2, encoded by the intronless GLUD2 gene on the X chromosome, which is primarily expressed in neural and testicular tissues, showing minimal to no expression in the liver. Studies have shown that GLUD1 is highly expressed in HCC patient samples and HepG2 cell lines. Silencing GLUD1 reduces HepG2 cell proliferation and induces apoptosis through the mitochondrial pathway. Knocking down GLUD1 triggers mitochondria-mediated apoptosis in HCC cells, thereby inhibiting their proliferation [237]. However, other studies have reported conflicting findings, showing that both GLUD1 protein and mRNA levels are downregulated in HCC compared to normal liver tissue. Overexpression of GLUD1 significantly inhibits HCC cell proliferation, migration, and tumor growth in vivo and in vitro, whereas GLUD1 knockdown promotes HCC progression. The underlying mechanism may involve GLUD1 overexpression altering HCC cell metabolism, leading to excessive ROS production and oxidative stress, which activates the p38/JNK/MAPK signaling pathway and induces mitochondrial apoptosis in HCC cells [238]. Given these conflicting results, the role of GDH in HCC remains controversial and requires further investigation to clarify its specific mechanisms. The dual nature of GDH in promoting or inhibiting HCC progression suggests that it may function differently depending on the cellular context or stage of tumor development, highlighting the need for deeper mechanistic studies.

Metabolites in glutamine metabolism

Gln and its associated metabolites are crucial in the initiation and progression of HCC. Gln is an essential nutrient for cell growth and proliferation, and rapidly proliferating cells, such as cancer cells, have an increased demand for Gln [239, 240]. A serum-based metabolomics study using 1 H-NMR demonstrated that Gln levels are significantly elevated in patients with cirrhosis and HCC compared to healthy individuals [241]. Gln metabolism is crucial in the interactions between the tumor microenvironment (TME) and tumor cells. Gln deprivation can induce the proliferation and activation of Tregs, which have immunosuppressive functions [242, 243]. In the TME of HCC, the imbalance in Gln distribution between CD8 + T cells and cancer cells leads to immune dysfunction. Research has shown that using Gln metabolism inhibitors can promote the proliferation of CD8 + T cells in the HCC microenvironment and enhance the efficacy of PD-1 blockers, improving the overall effect of immunotherapy [244]. Moreover, Gln influences HCC progression by modulating signaling pathways such as mTOR. In HCC, CTNNB1 mutations lead to β-catenin translocation and transcription factor activation, inducing the transcription of glutamine synthetase (GS). Elevated GS expression catalyzes the conversion of more glutamate into Gln, which in turn activates p-mTOR, promoting the proliferation of HCC cells [245]. Downstream Gln metabolites, such as hydroxyproline, have also been shown to play a critical role in promoting the hypoxic response in HCC. Mechanistically, hydroxyproline inhibits HIF1α hydroxylation and prevents its binding to tumor suppressor proteins during hypoxia, leading to increased HIF1α expression and further influencing HCC progression (Fig. 7) [243]. These findings highlight the multifaceted role of Gln metabolism in HCC, from supporting cancer cell growth to affecting immune responses within the TME, offering potential therapeutic targets for disrupting Gln metabolism to improve cancer treatment outcomes.

Fig. 7.

Gln metabolism in HCC

The figure depicts a simplified overview of the Gln metabolic pathways and associated signaling cascades. Glutamate-cysteine ligase (GCL) and cysteine (Cys) play important roles in cellular metabolism, and the TCA cycle is central to energy production. In HCC, the HGF/MET pathway stimulates glycolysis (Warburg effect) by activating glutaminase 1 (GLS1), promoting HCC cell proliferation. GLS1 further enhances cell proliferation through the Akt/GSK3β/CyclinD1 pathway. The SMYD2 protein reprograms Gln metabolism by regulating the c-Myc/GLS1 axis, driving HCC cell growth. Conversely, GLS2 inhibits HCC cell migration by promoting the maturation of miR-34a and suppressing Snail expression. The downregulation of glutamate dehydrogenase (GDH) leads to excessive ROS production and oxidative stress in HCC cells, which activates the P38/JNK/MAPK signaling pathway, inducing mitochondrial apoptosis and promoting cell death.

Drugs and clinical trials

Significant progress has been made in targeting Gln metabolism for cancer therapy, including approaches such as Gln deprivation, metabolic enzyme inhibitors, and transport protein inhibitors. GLS has emerged as an important target in tumorigenesis, and growing evidence suggests that inhibiting GLS may provide a promising strategy for selectively blocking tumor cell growth [246]. Some have already been applied in clinical trials (Table 1). GLS inhibitors, such as DON, BPTES, 968, CB-839, CPD23, and UPGL00004, have shown potential antitumor effects in Gln-dependent cancers [247]. For instance, GLS deficiency or treatment with the GLS-specific inhibitor BPTES was found to attenuate tumor progression and prolong survival in Myc-driven HCC mouse models [223]. It has also been shown that BPTES can suppress the growth of cancer cells in multiple tumor models, such as renal cell carcinoma (RCC) [248], breast cancer [249], and glioblastoma [250]. However, BPTES has poor solubility, limiting its clinical use. To overcome this limitation, a variety of BPTES derivatives, including CB-839, have been developed by researchers to enhance solubility. CB-839 has undergone evaluation in multiple Phase I-II clinical trials for the treatment of both hematologic malignancies and solid tumors [251]. Despite its promising vitro efficacy against triple-negative breast cancer (TNBC), CB-839 alone has shown limited therapeutic effects. While it exhibited good inhibitory effects on TNBC cells in vitro, it only achieved a 61% tumor inhibition rate in patient-derived TNBC xenograft models [252]. Similarly, CB-839 monotherapy has demonstrated suboptimal antitumor effects in HCC [253]. Consequently, CB-839 is increasingly being studied in combination therapies. For example, CB-839 combined with CDK4/6 inhibitors showed good efficacy in human esophageal squamous cell carcinoma [254]. Additionally, traditional Chinese medicine has attracted attention as part of targeted therapy strategies. Dihydroartemisinin (DHA) induces oxidative stress in cancer cells by increasing intracellular ROS. Combining GLS inhibitors with DHA significantly elevates intracellular ROS levels and decreases GSH content, exhibiting synergistic antitumor effects in HCC [255]. This also suggests potential therapeutic strategies for targeted therapy combining Chinese and Western medicine. Progress has also been made in developing inhibitors targeting Gln transport proteins. For example, Berberine inhibits HCC cell proliferation by reducing Gln uptake through suppression of c-Myc-induced SLC1A5, a key Gln transporter [256]. V-9302, a competitive small-molecule antagonist of transmembrane Gln flux, selectively inhibits SLC1A5 in a concentration-dependent manner. V-9302 suppresses cancer cell growth and proliferation, increases oxidative stress, and induces cell death. In vivo, V-9302 inhibited tumor growth in xenograft models, enhancing the sensitivity of Gln-dependent HCC cells to GLS inhibitor CB-839 by inducing ROS production and promoting apoptosis [253, 257]. Thus, dual inhibition of Gln transport protein SLC1A5 and GLS offers a novel potential therapeutic strategy for treating Gln-addicted HCC. These findings suggest that targeting Gln metabolism via inhibitors of GLS and Gln transport proteins like SLC1A5 may provide effective strategies for treating HCC, especially in combination with other therapies.

In conclusion, Gln metabolism undergoes significant alterations in HCC. Enzymes, metabolites, and transport proteins related to Gln metabolism hold potential as biomarkers for HCC. The reprogramming of Gln metabolism supports HCC cell growth and survival by providing essential carbon and nitrogen sources, as well as antioxidants, while activating signaling pathways such as mTORC to promote cell proliferation. Modulating Gln metabolism has become a potential therapeutic approach for treating HCC. Numerous studies indicate that changes in Gln metabolism not only contribute to the development and progression of HCC but also drive metastasis and drug resistance. However, the efficacy of inhibiting Gln metabolism alone appears to be limited. Combining Gln-targeted therapies with other treatment strategies may provide a more effective and promising therapeutic approach for HCC patients. This integrated approach could potentially address the challenges posed by tumor resistance and metastasis, leading to improved patient outcomes.

Metabolism of other amino acids

Serine metabolism

Serine (Ser), also known as β-hydroxyalanine, is a neutral aliphatic amino acid with a hydroxyl group and is classified as a non-essential amino acid for humans. It plays a critical role in glycogen storage in the liver and muscles, and in promoting cell proliferation. Ser is also involved in fat and fatty acid metabolism, muscle growth, and central nervous system function [258, 259]. Its biosynthetic pathway (SSP) involves several key steps: the glycolysis intermediate 3-phosphoglycerate (3-PG) is converted by phosphoglycerate kinase (PGK) into 3-phosphohydroxypyruvate (pPYR) with the help of phosphoglycerate dehydrogenase (PHGDH) and NAD+. pPYR is then transaminated by phosphoserine aminotransferase (PSAT) to generate phosphoserine (pSer), while also converting glutamate into α-KG, which enters the TCA cycle. pSer is subsequently dephosphorylated by phosphoserine phosphatase (PSPH) to produce Ser. Serine is further converted to glycine by serine hydroxymethyltransferase (SHMT), contributing to the folate pool, which supports nucleotide synthesis and the methylation of homocysteine to form methionine [260]. Additionally, 3-PG can reversibly generate 2-phosphoglycerate (2-PG) through the action of phosphoglycerate mutase (PGAM) (Fig. 8A). Research has shown that Ser metabolism is dysregulated in many tumor cells. Some tumor cells enhance Ser synthesis via glycolysis intermediates to support amino acid transport, nucleotide synthesis, redox homeostasis, and folate metabolism, thereby promoting cancer cell proliferation [261, 262]. In these pathways, PHGDH, which serves as the initial rate-limiting enzyme in the SSP pathway, is found to be elevated in several cancer types, such as breast cancer and melanoma [263, 264]. In glucose or glutamine-deprived environments, cancer cells significantly activate c-Myc-mediated SSP, increasing glutathione (GSH) synthesis, cell cycle progression, and nucleotide production, all of which are crucial for cell survival and proliferation [265]. In HCC, inactivation of PHGDH can inhibit SSP, reducing the production of α-KG, Ser, and NADPH, while increasing ROS levels, which induces apoptosis in HCC cells after treatment with sorafenib. The PHGDH inhibitor NCT-503, when combined with tyrosine kinase inhibitors (TKIs) such as sorafenib and lenvatinib, can effectively inhibit HCC growth and overcome TKI resistance [266]. However, some studies have reported that despite elevated Ser levels in HCC tissues, PHGDH mRNA and protein levels are significantly downregulated [267]. Mechanistic research suggests that the elevated Ser levels in HCC may result from the arginine methylation of PHGDH at residue R236 by protein arginine methyltransferase 1 (PRMT1), which enhances the catalytic activity of the enzyme. Inhibiting PHGDH methylation effectively suppresses HCC cell growth, indicating that PRMT1-mediated PHGDH methylation could be a potential therapeutic target in HCC.

Fig. 8.

Other amino acid metabolism processes, including serine, arginine, and methionine. A Schematic of the serine biosynthetic pathway. 3-phosphoglycerate (3-PG) is oxidized by PHGDH to 3-phosphohydroxypyruvate (pPYR), transaminated by PSAT to phosphoserine (pSer), and dephosphorylated by PSPH to yield serine. Serine is converted to glycine by SHMT, providing one-carbon units for nucleotide biosynthesis and the methionine cycle. 3-PG also reversibly interconverts with 2-phosphoglycerate (2-PG) via PGAM. 3-PG also reversibly interconverts with 2-phosphoglycerate (2-PG) by PGAM. B Schematic of the urea cycle. Citrulline formed from carbamoyl phosphate and ornithine in the mitochondria is exported to the cytosol and converted to arginine by ASS1 and ASL, consuming aspartate and releasing fumarate. Arginine hydrolysis by arginase yields urea and regenerates ornithine, which re-enters the mitochondria to maintain the cycle. C Schematic of the methionine cycle. Methionine Methionine is converted to S-adenosylmethionine (SAM), which donates methyl groups to form S-adenosylhomocysteine (SAH). SAH is hydrolyzed to homocysteine (Hcy), which is remethylated to methionine through folate- or betaine-dependent pathways. Methionine adenosyltransferase (MAT1A), catalyzing the first step of SAM synthesis, is frequently downregulated in liver cancer, highlighting its role as a metabolic tumor suppressor

In the process of drug development, targeting the serine metabolism pathway, particularly PHGDH inhibitors, has become a research focus. One compound, CBR-5884, inhibits PHGDH by interacting with its substrates NADH and 3-phosphoglycerate (3-PG) in a non-competitive, time-dependent manner, destabilizing the enzyme’s tetrameric form and converting it into a dimer. CBR-5884 effectively blocks serine de novo synthesis and serves as a potent PHGDH inhibitor [268]. However, CBR-5884 shows instability in mouse plasma, indicating the need for further optimization. Pacold et al. identified two potent PHGDH inhibitors, NCT-502 and NCT-503, through high-throughput screening [269]. These compounds exhibit favorable absorption, distribution, metabolism, and excretion (ADME) profiles. To validate target binding in cells, researchers used MDA-MB-231 cells engineered to stably express full-length human PHGDH (MDA-MB-231-PHGDH) and found that NCT-502 treatment significantly reduced intracellular serine and glycine levels without affecting other amino acids except aspartate. This demonstrated NCT-502’s strong target-binding effects. Additionally, Ixocarpalactone A, a natural compound derived from Physalis ixocarpa, directly binds to PHGDH and shows notable inhibitory activity. Ixocarpalactone A specifically targets and inhibits the proliferation of cancer cell lines exhibiting high PHGDH expression, including SW1990, MCF-7, and HeLa, while showing minimal cytotoxicity to normal human cells such as LO2, L929, and HPDE6-C7. Compared to previously discovered PHGDH inhibitors, Ixocarpalactone A exhibits high specificity and low toxicity, making it particularly promising for clinical translation, especially in pancreatic cancer [270]. These findings underscore the growing potential of PHGDH-targeted inhibitors as therapeutic agents in cancers that depend on serine metabolism, with ongoing research aimed at improving the efficacy and stability of these compounds.

Arginine metabolism

Arginine (Arg) is considered an essential amino acid for children, while it is conditionally essential for adults. As a functional basic amino acid, Arg is actively involved in numerous intracellular physiological activities and plays an essential regulatory role. In the human body, Arg participates in the urea cycle (also known as the ornithine cycle), promoting the conversion of toxic ammonia into non-toxic urea, thus reducing blood ammonia levels. In addition to being a component of proteins, Arg serves as a key substrate in several biochemical pathways, including nitric oxide (NO) production, creatine synthesis, and polyamine generation [271, 272]. Argininosuccinate synthase 1 (ASS1) is the rate-limiting enzyme in the urea cycle, catalyzing the condensation of citrulline and aspartate to form argininosuccinic acid, the precursor of Arg. This precursor can be further converted into Arg and fumarate (Fig. 8B). Studies have shown that ASS1 is downregulated or lost in various cancers, including HCC, malignant melanoma, and prostate cancer [273]. In these cancers, the inability to synthesize Arg internally forces the cells to rely on extracellular Arg for growth, a phenomenon referred to as Arg auxotrophy. Tumors with ASS1 deficiency are particularly sensitive to therapeutic strategies that target extracellular Arg degradation, and Arg deprivation triggers apoptosis in these tumor cells [274]. Therefore, ASS1 deficiency serves as both a prognostic biomarker and a predictor of sensitivity to Arg deprivation therapies [273]. Research has shown that ASS1 may act as a tumor suppressor in HCC. Stable silencing of ASS1 promotes the migration and invasion of HCC cells, while its overexpression inhibits metastasis in both in vitro and in vivo models. Mechanistically, overexpression of ASS1 inhibits the progression of HCC by inactivating the ^Ser727pSTAT3 signaling pathway and suppressing the expression of differentiation inhibitory factor 1 (ID1) [275]. Interestingly, despite the suppression of Arg synthesis in HCC, high levels of Arg are still observed in tumor tissues, indicating its essential role in tumor progression [276]. To explain this paradox, Mossmann et al. found that Arg acts as a second messenger-like molecule in tumors, regulating intracellular metabolism to promote HCC growth. High Arg levels bind to the regulatory factor RBM39, controlling the transcription and expression of metabolic genes. RBM39 enhances the expression of asparagine synthetase (ASNS), which increases asparagine production. Asparagine, in turn, further enhances the uptake of extracellular Arg, creating a positive feedback loop that maintains high Arg levels in HCC and drives pro-cancer metabolic processes [277]. These findings underscore the complex role of Arg in HCC progression, both as a nutrient and as a signaling molecule, making it a promising target for therapeutic interventions.

The loss of ASS1 in some tumor cells leads to a significant increase in their dependency on exogenous Arg. Recent studies have confirmed the efficacy of Arg depletion in various ASS1-deficient xenograft tumor models, including HCC, melanoma, small-cell lung cancer (SCLC), and pancreatic cancer [278–280]. Therefore, Arg deprivation is considered a novel anti-metabolic strategy for treating Arg-dependent cancers, and some approaches have already been applied in clinical trials (Table 1). Arginine deiminase (ADI) is an Arg-degrading enzyme isolated from Mycoplasma, which specifically converts extracellular Arg into citrulline without degrading other amino acids, thereby inhibiting the growth of Arg-dependent tumors such as melanoma and HCC. However, ADI suffers from a short half-life in mouse models, limiting its therapeutic efficacy. The pegylated form, ADI-PEG20 (Pegargiminase), was developed to improve its pharmacokinetics, offering a longer circulating half-life, reduced immunogenicity, and greater bioavailability, showing better efficacy in melanoma and HCC xenograft mouse models [273, 278]. Currently, ADI-PEG20 is in late-stage clinical development for various cancers, including HCC and malignant pleural mesothelioma (MPM). For cancers with reduced ASS1 expression, ADI-PEG20 as a monotherapy shows promising therapeutic potential. However, in cancers with normal or elevated ASS1 expression, ADI-PEG20 must be combined with other systemic therapies to achieve synergistic effects. For instance, in an open label, single arm, multicenter phase I clinical trial for acute myeloid leukemia (AML), ADI-PEG20 combined with Venetoclax and Azacitidine determined a recommended dose and demonstrated potential therapeutic efficacy, providing initial pharmacodynamic data and supporting further research [281]. Moreover, a phase Ib clinical trial indicated that ADI-PEG20 combined with the PD-1 antibody pembrolizumab exhibited good tolerability and safety in treating patients with advanced solid tumors [282]. Thus, combination therapies seem to outperform monotherapies in terms of therapeutic outcomes. Several trials combining ADI-PEG20 with other drugs are either ongoing or planned, as shown in Table 1. These clinical studies demonstrate that Arg-targeted therapies are advancing towards combination treatments, and there is growing anticipation that Arg-targeted therapies will soon become a standard option in cancer treatment.

Methionine metabolism

Methionine (Met) is classified as an essential amino acid required for human health and must be obtained through dietary intake. As a central component of one-carbon metabolism, Met serves as a critical link between the folate cycle and the transsulfuration pathway (Fig. 8C) [283]. Clinically, Met is mainly used for the treatment of chronic hepatitis and fatty liver disease due to its ability to promote fat metabolism in the liver. Changes in components of Met metabolism can significantly influence pathological states in chronic liver diseases. Cellular metabolism is highly dependent on Met, and tumor growth is closely tied to intracellular Met levels [284]. SIRT4, a highly conserved NAD+-dependent deacetylase, is involved in regulating post-translational modifications of proteins [285]. The mTORC1 signaling pathway controls metabolic processes in tumor cells by promoting the activation of multiple transcription factors, notably the oncogene c-Myc [286]. In HCC, there exists an abnormal mTORC1-c-Myc-SIRT4 axis that modulates cellular metabolism in a Met-dependent manner. Inhibition of SIRT4 expression leads to the activation of the mTORC1-c-Myc pathway, resulting in Met metabolism reprogramming, which triggers high tumor cell proliferation and impacts HCC progression [287]. In mammals, Within the folate cycle, Met is transformed into S-adenosylmethionine (SAM) through the catalytic action of methionine adenosyltransferase (MAT). The MAT gene family includes MAT1A, MAT2A, and MAT2B, encoding the protein products MATα1, MATα2, and MATβ, respectively. MATα1 and MATβ are not co-expressed, and MAT2B encodes the regulatory subunit of MATα2, controlling its activity. Research has shown that MAT gene dysregulation plays a crucial role in liver disease, with the switch from MAT1A to MAT2A/MAT2B closely associated with the progression of chronic liver disease and HCC [288]. MAT1A is primarily expressed in the liver and maintains hepatocyte differentiation. Its expression is reduced in the majority of individuals with cirrhosis, alcoholic hepatitis, during cellular dedifferentiation, and in cases of HCC. In contrast, MAT2A and MAT2B are typically expressed in non-parenchymal cells of the liver and other extrahepatic tissues, but their expression is induced in HCC [289–292]. In HCC, MAT1A downregulation leads to increased oxidative stress, hepatic stem cell proliferation, and genomic instability [292]. Overexpression of MAT1A can increase SAM levels, promote apoptosis, and inhibit the expression of angiogenesis-related genes, thus suppressing tumor growth [288]. Specifically, MAT1A-knockout (MAT1A-KO) mouse models exhibit low GSH levels, increased expression of CYP2E1, oxidative stress, and mitochondrial dysfunction. CYP2E1 is a key enzyme responsible for metabolizing hepatotoxins such as CCl4 and plays a critical role in ROS production. Thus, MAT1A-KO mice are more sensitive to hepatotoxin-induced liver damage [293]. Hepatic stem cells, which are normally quiescent in the adult liver, only proliferate in response to chronic liver damage or in experimental carcinogenesis models. Rountree et al. found that with aging, MAT1A-KO mice showed expansion of hepatic stem cells, which increased oncogenic signals such as Kras and Survivin, and these cells had tumorigenic potential in vivo [294]. Genomic instability—marked by mutations, duplications, deletions, and replication errors within cancer cells—is believed to represent an initial step in the progression of cancer [295]. Dysregulation of MAT genes is closely related to genomic instability, and early disturbances in MAT protein function can influence cancer progression. MAT1A regulates DNA methylation via SAM, and MAT1A deficiency results in DNA hypomethylation, leading to genomic instability. Furthermore, alterations in MAT protein activity and global DNA hypomethylation are prognostic biomarkers for human HCC. Overexpression of MAT1A in the Huh7 cell line increases SAM levels, promotes apoptosis, inhibits tumor cell growth, and reduces the expression of angiogenesis-related genes [294]. Previous studies have shown that several miRNAs (e.g., miR-664, miR-485-3p, and miR-495) that negatively regulate MAT1A are upregulated in HCC. Silencing these miRNAs in Hep3B and HepG2 cell lines induces MAT1A expression and promotes apoptosis. In mouse HCC orthotopic models, siRNA treatments targeting these miRNAs reduced tumor growth, invasion, and metastasis [296]. Therefore, enhancing endogenous MAT1A expressions in HCC cells represents an attractive therapeutic strategy warranting further investigation. SAM acts as the primary methyl donor in many biochemical reactions and as a precursor for polyamine synthesis [297]. In tumor cells, alterations in MAT expression impair SAM-related biosynthesis in the liver. Guo et al. discovered the existence of a Met-MAT2A-SAM axis in cancer cells, which plays a vital role in tumorigenesis by providing SAM as a substrate for abnormal protein, DNA, and RNA methylation [298]. Exogenous SAM can inhibit HCC progression by restoring normal levels of SAM. Pharmacological concentrations of SAM and its metabolite MTA promote apoptosis in HCC cells while sparing normal cells [299]. SAM has also shown clinical value in improving the prognosis of patients with advanced tumors. However, the efficacy of SAM still needs further exploration through randomized prospective clinical trials [300].

The research on several amino acids outlined above has only preliminarily revealed their roles in the metabolic activities that contribute to the development of HCC cells. However, due to the highly complex and dynamic nature of tumor metabolism, achieving more effective treatments requires deeper exploration of the molecular mechanisms underlying these metabolic pathways. Many key issues remain unresolved and need to be elucidated and clarified through further studies. These include understanding how various amino acid metabolism pathways interact with one another, identifying the precise regulatory networks that control metabolic reprogramming in HCC, and determining how metabolic changes contribute to drug resistance, tumor proliferation, and metastasis. Additionally, further research is needed to explore potential therapeutic targets within these pathways and to develop novel strategies for overcoming the metabolic adaptability of HCC cells. Only through comprehensive investigations into these unresolved mechanisms can we improve therapeutic interventions and provide better outcomes for patients with HCC.

Conclusion

HCC, one of the deadliest malignancies globally, is often difficult to detect in its early stages and remains a major public health challenge. While surgery and conventional chemotherapy have improved patient prognosis to some extent, their overall efficacy remains limited, particularly for advanced HCC patients. Similar to other malignancies, the occurrence and progression of HCC is a complex, multi-step, and multi-factorial process. Among these factors, metabolic alterations play a crucial role in the transformation of normal cells into cancer cells. The Warburg effect, first proposed and explained by Otto Warburg, provided new insights into the metabolic changes associated with tumorigenesis. As research has progressed, metabolic reprogramming has become recognized as a hallmark of many cancers, including HCC. In HCC patients, numerous metabolic-related genes are consistently dysregulated. Therefore, investigating the metabolic alterations and gene regulation involved in HCC development and restoring these abnormal metabolic pathways are critical for exploring new therapeutic strategies and improving patient outcomes. Since the 20th century, growing evidence has confirmed that HCC cells’ dependency on amino acids extends beyond protein synthesis to energy requirements. The utilization of key amino acids, such as aromatic amino acids, branched-chain amino acids, and glutamine, plays an essential role in HCC progression. Amino acid metabolic reprogramming promotes biological processes in HCC, including proliferation, apoptosis, migration, drug resistance, and immune evasion. Additionally, alterations in metabolic products, transport proteins, transcriptional regulators, and signaling molecules involved in amino acid metabolism further impact HCC progression. These changes have a significant influence on patient prognosis and survival. The challenge of preventing or disrupting these processes to achieve “tumor-bearing survival” has become a central focus and difficulty in current HCC research. Although amino acid metabolism has been recognized as a key driver of HCC progression, limitations remain. Current studies primarily focus on a few well-known pathways, such as Gln, BCAAs and AAAs, while the broader spectrum of amino acid metabolism and its dynamic interplay with other metabolic networks are not fully understood. Moreover, most existing evidence is derived from molecular or in vitro studies, which cannot fully recapitulate the complexity of the tumor microenvironment. A comprehensive understanding of how these metabolic processes relate to tumor development is essential for developing new therapeutic drugs targeting tumor energy metabolism and promoting their clinical application. Such an approach could potentially open new avenues for more effective treatments for HCC.

Abbreviations

HCC

Hepatocellular carcinoma

AAAs

Aromatic amino acids

Trp

tryptophan

Tyr

tyrosine

Phe

phenylalanine

Phe

Phenylalanine

Leu

leucine

Ile

isoleucine

Val

valine

Glu

glutamate

Ala

alanine

Arg

Arginine

Gln

glutamine

PAH

phenylalanine hydroxylase

5-HT

5-hydroxytryptamine (serotonin)

Kyn

kynurenine

AADC

aromatic L-amino acid decarboxylase

TDO

tryptophan 2,3-dioxygenase

IDO1

indoleamine 2,3-dioxygenase 1

IDO2

indoleamine 2,3-dioxygenase 2

AhR

aryl hydrocarbon receptor

QuinA

quinolinic acid

TCA

tricarboxylic acid

TAT

tyrosine aminotransferase

GSTZ1

glutathione S-transferase zeta 1

FAH

fumarylacetoacetate hydrolase

MAA1

maleylacetoacetate isomerase

FAA

fumarylacetoacetate

BCATs

branched-chain amino acid aminotransferases

BCKAs

branched-chain α-keto acids

α-KG

α-ketoglutarate

BCKDH

branched-chain α-keto acid dehydrogenase

BCKDK

branched-chain α-keto acid dehydrogenase kinase

KIC

α-ketoisocaproic acid

KIV

α-ketoisovaleric acid

KMV

α-ketomethylvaleric acid

GLS

glutaminase

GSH

glutathione

GDH

glutamate dehydrogenase

Authors’ contributions

BW and LW designed and guided the review. RZ wrote and edited the manuscript. GL, YL and LL helped with reference collection. YL and RZ drew the figures. All authors read and approved the final manuscript.

Funding

This study was supported by grants from the National Natural Science Foundation of China (82003924), the 2023 Shanghai “Rising Stars of Medical Talents” Youth Development Program (Youth Medical Talents–Clinical Pharmacist Program), the Jing’an District Excellent Youth Program of the Health System (2024YQ02), the Jing’an District Health Research Project (2019QN02) and the Jing’an District Discipline Construction Project (Key Discipline, 2024ZD01).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors have read and approved the final version of this manuscript.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Competing interests

The authors declare no competing interests.