Introduction

Amino acids, the basic units of proteins, are widely involved in the formation of energy, synthesis of macromolecules, and signal transduction in cells. They are essential for the survival of cancer cells. Amino acid metabolism is an important metabolism process in cancer cell and has attracted the extensive research attention, particularly the metabolism of non-essential amino acids. Among them, the most studied non-essential amino acid is glutamine that contributes to cancer cell proliferation, invasion, and migration. Glutamine is the highest content of amino acids in plasma, but many cancer cells easily produce glutamine addiction due to the high demands for nutrient, especially in cancer cells that enhanced myelocytomatosis oncogene (MYC) protein expression [1]. Therefore, glutamine metabolism has become important targets for diagnostic imaging and treatment of cancers [2]. With the development of clinical research, investigators have gradually enhanced the study of asparagine to provide a vital theoretical basis for its use as a cancer-treatment target.

Asparagine, a non-essential amino acid, can be produced by de novo synthesis in addition to being obtained from food. Two enzymes are involved in asparagine metabolism: asparagine synthase (ASNS), which catalyses glutamine- or ammonia-dependent asparagine synthesis from aspartate, and asparaginase (ASNase), which hydrolyses asparagine to aspartate. Aspartate is mainly generated in the mitochondria through the respiratory chain [3]. In humans, ASNS is expressed in several organs, and the highest levels of ASNS activity are observed in the pancreas. While ASNase is expressed in only a few human organs, such as the liver and kidneys. Numerous studies indicated that asparagine metabolism is essential for the growth and development of cancer cells [4]. Briefly, asparagine metabolism in cancers mainly refer to cancer cells upregulate ASNS expression and further catalyses synthesis of asparagine via various signaling pathways in order to meet the needs of growth, and the mechanism of asparagine involved in cancer cells growth and metastasis.

Nutritional restrictions are often used against cancer because of the high basal metabolic rate and nutritional requirements of cancer cells [2]. One of the most significant therapeutic strategies is asparagine restrictions. For cancer cells, the amount of asparagine synthesised by themselves cannot meet their need for asparagine; therefore, they are more sensitive to exogenous asparagine than normal cells. Clinically, ASNase has successfully suppressed leukaemia by specifically reducing circulating asparagine levels [5]. However, ASNase is not as effective for treating other solid cancers. Investigators have explored the reasons for the poor efficacy of ASNase. Because asparagine is obtained from circumstance and ASNS-dependent de novo synthesis, different cells show different sensitivities to ASNase owing to different intracellular levels of ASNS expression. While ASNS protein expression levels are closely related to many regulators in the cells. Different cancer cells have unique metabolic characteristics, and they specifically adjust asparagine metabolism to meet their energy and nutrient requirements [6]. Therefore, a comprehensive understanding of the metabolism and role of asparagine has important clinical implications and potential applications [7]. This will help increase the therapeutic efficacy of ASNase during cancer therapy, search for more effective treatment strategies and diagnostic approaches, and reduce the risk of side effects.

Physiological functions of asparagine

The asparagine-dependent metabolism of the nutrients

In proliferating cells, asparagine is one of the least abundant non-essential amino acids [4]; however, it is essential for cell survival. A previous study indicated that the main purpose of mitochondrial respiration was to synthesise asparagine [8]. With the increasing research on asparagine, the role of it is not just limited to as the substrates for protein synthesis. As early as 1883, Schulze and Bosshard discovered a tendency for the spontaneous deamidation of asparagine under mild conditions. However, this process does not require catalytic enzymes. It is primarily determined by the amino acid sequence surrounding asparagine and is governed by multiple layers in the protein interior. When some amino acids are altered, they may cause deamidation of key asparagine molecules around them, making asparagine a regulator of protein turnover [9]. There is a negative relationship between the asparagine content and protein lifetime. However, some studies have suggested that spontaneous deamidation of asparagine generates an isoaspartate residue that hampers protein function and induces disorders associated with senescence [10]. Sequence- and structure-based methods can detect asparagine deamidation in proteins [11]; thus, we can predict the function of proteins through these methods.

Asparagine also plays an important regulatory role in the metabolism of other nutrients. Compared with other amino acids, asparagine can activate the mammalian target of rapamycin complex 1 (mTORC1) through ADP-ribosylation factor 1 (ARF1) in a Rag GTPase-independent manner [12]. mTORC1 phosphorylates ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) when stimulated by cell growth signals [13]. S6K1, one of these targets, mediates the phosphorylation of carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotatase (CAD) at Ser1859 which catalyses the de novo synthesis of pyrimidine [14]. Moreover, asparagine can directly offer γ-nitrogen for the biosynthesis of purine and pyrimidine [15]. Phosphorylated 4E-BP1, another mTORC1 target, blocks its binding to eIF4E, enabling it to form the eIF4E complex required for initiating protein translation [16].

The role of asparagine has been preliminary studied in adipose tissue as well. Brown and beige adipocytes primarily consume energy generated by the oxidation of fatty acids and glucose in the form of heat [17]. When brown adipocytes were cultured in a medium containing asparagine, the expression levels of lipogenic and thermogenic genes increased compared to the control group. In acute cold exposure experiments, an improvement in cold resistance was observed in mice after supplementation with asparagine. In contrast, when treated with ASNase, acute cold stimulation induced hypothermia in mice. Further metabolomic analysis and isotope tracing showed that the levels of key enzymes and glycolytic intermediates were significantly increased. It has been proposed that glucose is the primary source of thermogenesis in the adipose tissue [18]. When adipocyte glucose transporters (such as glucose transported type 1 (Glut1), Glut4, hexokinase 2 (HK2), or pyruvate kinase (Pkm)) are knocked down, both thermogenesis and oxygen consumption are reduced in brown adipose tissue (BAT) [19]. Therefore, asparagine promotes adipocyte thermogenesis, at least in part, by increasing glycolysis. In addition, these regulatory mechanisms are involved in the mTORC1 signalling cascade [20] (Fig. 1).

Fig. 1
figure 1

Connections between asparagine and other nutrients. In addition to comprising the basic component of the protein-peptide chains, asparagine regulates the intake of amino acids by serving as an amino acid exchange factor and protein turnover by serving as a regulator. Asparagine also plays a role in the synthesis of nucleic acid molecules, the glycolysis process, and heat production in adipocytes through mTORC1 signalling cascades

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Asparagine is important during glutamine deprivation

In addition to maintain the most basic physiological metabolism, asparagine is particularly crucial when cells are starved for nutrients, especially glutamine. Glutamine is required for de novo asparagine synthesis. It is both a carbon and nitrogen source for asparagine. Glutaminase catalyses the hydrolysis of glutamine to glutamate and ammonia. Glutamate then enters the tricarboxylic acid cycle (TCA cycle) and the respiratory chain to generate aspartate in the mitochondria, which subsequently enters the cytoplasm through transporters. Finally, ASNS converts aspartate to asparagine using glutamine as a nitrogen donor [6]. Glutamine is a non-essential amino acid that plays an important role in cell proliferation and survival, and is involved in the synthesis of other nutrients and various cellular activities [21]. Glutamine deprivation induces cell apoptosis. And it was confirmed that the percentage of living cells was significantly increased when citrate synthase (a TCA cycle enzyme) was knocked down [4]. Citrate synthase (CS) catalyses the formation of citrate from oxaloacetate and acetyl-CoA. This pathway is blocked when CS is inhibited, leading to the transition of oxaloacetate to aspartate and asparagine. This conversion rescues the glutamine-induced apoptosis. Recent studies have shown that glutaminase 1 assembles into a filament-like shape after glutamine deprivation. This shape possesses high activity and substrate-binding affinity, leading to a reduction in intracellular glutamine and, subsequently, intracellular asparagine. Several types of mitogenome-encoded protein (MEPs) synthesis pathways rely on asparagine. Therefore, MEPs will also lack, which further impair electron transfer chain (ETC) function and trigger an outburst of mitochondria-derived reactive oxygen species (ROS) [22]. These signals also result in the intrinsic apoptosis of cells [23]. The addition of asparagine to the medium can restore cell proliferation by preventing ROS burst in long-term glutamine starvation cells, but not alanine, proline, glutamate and aspartate [4, 15]. Glutamine also regulates angiogenesis through multiple mechanisms. The proliferation of endothelial cells (ECs) and vessel sprouting are impaired when exogenous glutamine is not available. At this time, ECs rely on asparagine for proliferation [24]. Asparagine alone can partially rescue ECs defects under low glutamine conditions [25]. Together, these results suggest a critical role for asparagine in cellular adaptation to glutamine deprivation [4]. Asparagine can also exchange extracellular amino acids as an amino acid exchange factor like glutamine. And cells preferentially utilise asparagine as an amino acid exchange factor [26]. Asparagine maintains cell life activities like glutamine, and also seemingly plays a more significant role compared to glutamine because the overconsumption of intracellular asparagine can influence cellular proliferation and induce cell apoptosis, even under glutamine-rich conditions (Fig. 2) [4].

Fig. 2
figure 2

The role of asparagine during cellular stress. Asparagine is synthesized under the catalytic action of ASNS using aspartate and glutamine as raw materials, in which glutamine serves as the carbon source as well as the nitrogen source. When cells are under stress due to the shortage of nutrients, ISR or ERS are initiated, which increases the production of asparagine to maintain cell growth and development by upregulating the expression of ASNS. When raw materials are deficient or the expression of ASNS fails to be activated, cells cannot synthesize sufficient asparagine, leading to apoptosis through ETC damage. Mito, Mitochondrion

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Adaptive responses to cellular stress

Asparagine is an important regulator of the stress response in cells. The integrated stress response (ISR) of cells is induced by the starvation of various nutrients, such as amino acids and proteins. Upon stimulation, uncharged tRNA binds to general control nonderepressible 2 (GCN2), leading to its dimerization and autophosphorylation. Activated GCN2 phosphorylates eukaryotic translation initiation factor 2 subunit α (eIF2α) to block the initiation of mRNA translation and globally inhibit protein translation. It is also possible to elicit endoplasmic reticulum stress (ERS), also known as the unfolded protein response (UPR). It is initiated by the activation of pancreatic ER kinase (PKR)-like ER kinase (PERK), then phosphorylates eIF2α [87]. Of these, asparagine was also included. Upon glutamine depletion, ASNS knockdown accelerates macropinocytosis. Inhibition of macropinocytosis and the ASNS gene alone reduced the growth rate of CRC cells, but the combination of both almost completely suppressed cancer growth. The combination group showed no observed effect on mouse weight [88]. This may be a promising novel strategy for the treatment of KRAS mutations.

In addition to KRAS mutations, p53 mutations are another common type of mutation in CRC, and patients with p53 mutations experience poor treatment outcomes. P53 participates widely in various anti-proliferative reactions as a cancer suppressor. A recent study revealed a novel association between asparagine and p53. Under physiological conditions, P53 can bind to ASNS and inhibit its expression, which regulates homeostasis between aspartic acid and asparagine. In turn, decreased asparagine is perceived by LKB1, which then activates MAPK and subsequently induces p53-dependent cell cycle arrest, thereby protecting the cells from apoptosis. Moreover, apoptosis of p53-null CRC cells increased in the absence of asparagine due to increased asparagine sensitivity [89]. Therefore, in CRC cells with p53 mutations, asparagine limitation provides drug therapy, making CRC cells more sensitive to radiotherapy [90].

These findings suggest that some critical genes that influence the development of CRC correlate with asparagine metabolism, thus providing further possibilities for the use of asparagine deficiency in the treatment of CRC.

ASNase therapy

ASNase, a chemotherapeutic agent targeting free asparagine, has been approved for cancer treatment and has achieved a certain degree of success in the clinical treatment of patients with leukaemia. The combination treatment with ASNase and vindesine, as well as combined treatment with ASNase and prednisone can induce remission in up to 90% of children with ALL. And ASNase may enhance the sensitivity of cancer cells to radiotherapy. ASNase is generally well tolerated by most patients, and few patients develop an anaphylactic reaction or anti-ASNase antibodies when they initially receive ASNase [91]. High ASNase activity in the blood can effectively prevent central nervous system relapse and improve prognosis. ASNase is an ideal chemotherapeutic drug even for infants with leukaemia. In one study, almost all children with ALL achieved a complete remission after ASNase treatment [92]. The treatment effect of ASNase depends on the asparagine consumption level as well as on the corresponding duration. Any residual asparagine can cause treatment failure or cancer recurrences [93]. Therefore, it is beneficial to combine therapeutic drug monitoring (TDM) with ASNase treatment [94]. In children with B-acute lymphoblastic leukaemia (B-ALL), the levels of asparagine in the plasma and bone marrow showed a strong correlation, whereas there was no significant correlation between the plasma and cerebrospinal fluid (CSF) [95, 96]. Depletion of asparagine in the CSF helps reduce central nervous system involvement; therefore, it can achieve the best monitoring effects to simultaneously measure asparagine in both plasma and CSF. However, the measurement of plasma asparagine concentrations is more frequently performed because it is difficult to obtain CSF. In addition to the direct measurement of asparagine, we can also determine ASNase activity to monitor asparagine depletion in the serum [97].

However, ASNase may introduce undesirable side effects such as thrombus, hypersensitivity, hyperglycemia, hypertriglyceridemia, acute pancreatitis, and hepatotoxicity [20, 92, 98, 99]. For decades, researchers continually improve ASNase structure, in order to increase treatment outcomes and reduce side effects. Native E. coli L-ASNase and pegaspargase are major components of ALL treatment regimens. At present, many modified asparaginases have appeared. For example, the recombinant L-ASNase from the genus Anoxybacillus possesses good thermal stability without glutaminase activity [100]. And L-ASNase GRASPA (®), which is encapsulated in red blood cells, is well tolerated and reduces the occurrence of allergic reactions and coagulation disorders [101]. However, almost all of these ASNase studies are based on leukaemia cells. If we want to use it for the treatment of other solid cancers, further in vitro and in vivo studies may be needed to evaluate its actual effectiveness in other cancers. Moreover, with the extensive study of asparagine, asparagine metabolism in cancer is gradually being unveiled. Thus, combination of ASNase with asparagine metabolism will achieve more precise treatment.

Discussion

Cancer is the second most common cause of death and a worldwide threat to human health. Clinical researchers are continuously seeking effective methods and medicine of treatments. Cancer cells have a relative nutrient deficiency because of their high metabolic rates. They often sustain their survival and development through various metabolic reprogramming processes [102], which are considered specific hallmarks of cancer [103]. First, these stress processes are considered to produce more glutamine [88]. However, an increasing number of studies have shown that cancer cell growth is dependent on asparagine. In mammals, asparagine is not broken down but is primarily involved in protein translation. Asparagine-mediated protein translation is necessary for the proliferation and migration of adaptive cells [75]. Interestingly, asparagine also regulates senescence. For example, during glutamine deficiency, p53-dependent senescence was reversed by asparagine supplementation [89].

Metabolomic analysis is a useful method for identifying clinically meaningful biomarkers and treatment targets and is widely used in the field of cancer research [104]. Metabolomic analysis have shown that asparagine is closely related to cancer progression and metastasis. Therefore, asparagine targeting has gradually become a promising strategy for cancer treatment. Depletion of circulating asparagine stimulates cells to initiate stress signalling cascades and upregulates ASNS gene expression. Although these stress responses eventually promote ASNS expression, there are different regulatory programs in response to nutritional stress response in cancer cells. By fully understanding the metabolic processes of asparagine in different cancers, we can select specific inhibitors to block these compensatory pathways. Then, the source of asparagine is further cut off, which is more effective in inhibiting cancer cells and improve clinical outcomes especially for solid cancers that are not sensitive to ASNase alone. There is much experimental evidence indicated these combined treatments particularly effective.

In the previous content, we have reviewed not only asparagine metabolism in several cancers but also in the case of certain gene mutations, which provide many targets for cancer therapy (Table 1). However, there are still some limitations. Including leukaemia, there was no clear evidence demonstrates ASNS expression levels predict the sensitivity of cancer cells to ASNase. Moreover, in human-derived breast cancer cell lines, we cannot conclude that ASNS promotes cancer progression by upregulating asparagine levels. And ASNS can influence lung cell invasiveness through an alternative pathway other than asparagine. There have been inhibitors that directly target ASNS [23]. Perhaps this allows us to confuse the relationship between ASNS and asparagine. But this also further illustrates the metabolism specificity of asparagine in cancer cells and adds depth to the discussion.

Table 1 Signal molecules and processes related to asparagine metabolism in various cancers
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Immunotherapy has become one of the most important therapeutic strategies for treating cancers, which has greatly promoted the progress of cancer treatment. The immune checkpoint blockade (ICB) based on monoclonal antibodies targeting immune checkpoint proteins and adoptive cell transfer (ACT) based on tumour-infiltrating lymphocytes or CAR T cells are frontline cancer immunotherapies. Recently, researchers have begun to relate asparagine and immunity. In the early stages of antigen stimulation, asparagine induces the transition of naïve CD8 + T lymphocytes to an active state by phosphorylating lymphocyte-specific protein tyrosine kinase (Lck) [105]. While for activated CD8 + T lymphocytes, asparagine restriction can enhance CD8 + T cell metabolic fitness and antitumoral functionality through the Nrf2-dependent stress response. In preclinical animal models, the combination of asparagine restriction with anti-PD-L1 antibodies displayed a better anti-tumour effect than the anti-PD-L1 monotherapy alone group [106]. These results suggested that Asn restriction is a promising and clinically relevant strategy to enhance cancer immunotherapy against multiple cancer types.

Conclusion

After decades of research on asparagine, its essential role in mammals cannot be ruled out. Asparagine is essential for cancer growth and development. It can participate in the metabolism of other intracellular nutrients via mTORC1 signaling cascade, maintaining the nutrient demand of cancer cells, and promote cancer metastasis by influencing EMT pathway. Once asparagine is insufficient, cancer cells can activate ISR and ERS to upregulate the expression of ASNS to synthesize sufficient asparagine. Moreover, for different cancer cells, there are also different programs to regulate asparagine metabolism, but ultimately lead to increased expression of ASNS. Based on the importance of asparagine in cancer cells, ASNase targeting asparagine has been used for the treatment of leukaemia. However, the treatment effect of ASNase in other solid cancers is not good, mainly because of the mechanism of reprogramming asparagine metabolism. With the roles of asparagine in the physiological state and stress response are gradually explored, this limitation will hopefully be addressed in the future. By combining inhibition of ASNS or inhibition of targets that regulate ASNS with asparagine restriction, the level of asparagine will be greatly reduced and the growth of cancer cells will be inhibited. At present, there are two methods of asparagine restriction: dietary restriction and ASNase treatment. ASNase is being refined to improve efficacy and reduce side effects. In addition, the combination of asparagine restriction with radiotherapy and immunotherapy has also begun to become a new cancer treatment strategy. Although there is preliminary experimental evidence that revealed the efficacy of the combination therapy, more experimental data are needed to support it. In the future, asparagine remains an ideal target for the strategy of nutrient restriction.