Figure 1. Schematic representation of key metabolic pathways for the biosynthesis of NEAAs, the enzymes in each pathway, and the main functions of each AA
[35][78]. NEAAs are represented in blue and EAAs in red. Leucine (Leu), isoleucine (Ile), histidine (His), valine (Val), phenylalanine (Phe), threonine (Thr), methionine (Met), lysine (Lys), tryptophan (Trp), glutamine (Gln), alanine (Ala), aspartate (Asp), asparagine (Asn), arginine (Arg), tyrosine (Tyr), glutamate (Glu), cysteine (Cys), glycine (Gly), proline (Pro), and serine (Ser). 3-phospho-D-glycerate (3-PG), S-adenosylmethionine (SAM), homocysteine (HCys), glutathione (GSH), α-ketoglutarate (α-KGlu), tricarboxylic acid cycle (TCA), oxaloacetate (OAA), reactive oxygen species (ROS). D-3-phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase-1 (PSAT1), vacuolar protein sorting-associated protein-29 (VPS29), phosphoserine phosphatase (PSPH), serine hydroxymethyltransferase-1 (SHMT1), serine hydroxymethyltransferase-2 (SHMT2), S-adenosylmethionine synthase isoform type-1 (MAT1A), S-adenosylmethionine synthetase isoform type-2 (MAT2A), methionine adenosyltransferase 2 subunit beta (MAT2B), adenosylhomocysteinase (AHCY), cystathionine β-synthase (CBS), cystathionine γ-lyase (CTH), methionine synthase (MTR), betaine-homocysteine methyltransferase (BHMT), betaine-homocysteine methyltransferase-2 (BHMT2), S-adenosylmethionine decarboxylase (AMD1), spermidine synthase (SRM), 5′-methylthioadenosine phosphorylase (MTAP), methylthioribose-1-phosphate isomerase (MRI1), methylthioribulose 1-phosphate dehydratase (APIP), enolase-phosphatase (ENOPH1), 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase (ADI1), 2-oxo-4-methylthiobutanoate aminotransferase (KYAT1), phenylalanine hydroxylase (PAH), alanine aminotransferase-1 (GPT), alanine aminotransferase-2 (GPT2), glutaminase-1 (GLS1), glutaminase-2 (GLS2), glutamine synthetase (GLUL), glutamate dehydrogenase-1 (GLUD1), glutamate dehydrogenase-2 (GLUD2), aspartate aminotransferase-1 (GOT1), aspartate aminotransferase-2 (GOT2), asparagine synthetase (ASNS), asparaginase (ASRGL1), aspartylglucosaminidase (AGA), argininosuccinate synthase (ASS1), argininosuccinate lyase (ASL), ornithine aminotransferase (OAT), pyrroline-5-carboxylate reductase-1 (PYCR1), pyrroline-5-carboxylate reductase-2 (PYCR2), and δ-1-pyrroline-5-carboxylate synthase (ALDH18A1). Enzymes that participate in consecutive steps in a metabolic pathway are separated by “-” and enzymes that catalyze the same step in a metabolic pathway are separated by “/”.
The importance of Cys in tumor growth was first reported in 1936
[79]. In this study, Voegtlin et al. observed that a diet deficient in Cys/Met reduced tumor growth in mice with spontaneous breast cancer, and the addition of Cys abruptly stimulated tumor growth
[79]. Since Met can produce Cys, Met has usually been restricted in many studies evaluating the anticancer activity of Cys depletion/restriction
[21][27][33][34][59][61][68][69][70][79]. Intravenous parenteral nutrition with double Cys/Met restriction showed anticancer activity in rats with sarcoma
[69][70] and inhibited the cancer proliferation in mice xenografted with human glioma cells
[59].
Mechanistically, Cys restriction may induce anticancer activity by reducing the capacity of cancer cells to eliminate ROS. Cancer cells produce high levels of ROS, which may accumulate and produce cell death
[8]. Cancer cells rely on GSH to reduce these ROS levels
[50]. A dietary Cys restriction can decrease Cys plasma levels
[80], reduce GSH biosynthesis
[80][81], and increase the ROS levels in cancer cells
[80][81][82].
Since Cys is necessary for immune cells, Cys restriction may reduce the ability of the immune system to eliminate cancer cells. Cys is essential for T-cell activation and function
[83]. High CysS plasma levels have been associated with a higher probability of response to immune checkpoint inhibitors in patients with lung cancer
[84][85]. However, the negative effect of Cys restriction on the immune antitumor response is controversial, because other studies have suggested that Cys restriction can increase the antitumor immune response
[21][86].
Pharmacological approaches based on an enzymatic depletion of Cys and inhibition of Cys transporters support the idea that Cys restriction has potential for cancer therapy. These pharmacological interventions have been useful for understanding the possible mechanisms by which Cys restriction induces in vivo anticancer effects. In 2017, an optimized human cyst(e)inase enzyme was able to reduce the Cys and CysS plasma levels in mice and primates without causing toxicity
[87]. Cyst(e)inase has shown anticancer activity in mouse models of a variety of cancers, including prostate, breast, chronic lymphocytic leukemia, pancreas, lung, renal, melanoma, and ovarian cancer
[86][87][88][89][90][91][92]. Cyst(e)inase administration increases ROS levels, depletes the intracellular levels of GSH, and triggers ferroptosis in cancer cells
[87][88][89][90][91][92].
4.2. Serine
Serine (Ser) is synthesized from 3-phosphoglycerate (glucose metabolite) and Glu (nitrogen donor) through the de novo Ser synthesis pathway
[93]. In addition to being a proteinogenic AA, Ser plays an important role in one-carbon metabolism
[93][94][95]. Ser is the main source of carbon units in the folate cycle, which is mainly used for the synthesis of purines and pyrimidines and the conversion of HCys into Met. Ser is also used to produce Gly and provides the carbon skeleton for the synthesis of Cys through the transsulfuration pathway. It also has other important functions, such as the production of certain lipids, including ceramide and phosphatidylserine
[93][94][95].
Ser and Gly are easily interconverted by SHMT1/2 enzymes
[95]. Therefore, Gly is usually restricted in most dietary studies evaluating the anticancer activity of Ser limitation. Dietary Ser/Gly can reduce the Ser and Gly levels in plasma
[96] and tumors
[97]. Although both AAs can be synthesized by human cells, cancer cells may depend on an external supply of these AAs to keep their high proliferative demands.
Mechanistically, dietary Ser/Gly restriction can induce anticancer activity by restricting two important building blocks in biosynthesis. The new cancer cells created during tumor growth need new proteins, nucleic acids, and specific lipids; these processes require the synthesis or acquisition of sufficient levels of these two AAs.
4.3. Glycine
Gly is an NEAA that can be synthetized from Ser. Gly is essential for protein synthesis. Collagen, which is the most abundant protein in the human body (30–40% of total body protein), contains approximately 33% of Gly
[98]. This AA also acts as an inhibitory neurotransmitter
[24]. Gly can also be used for the synthesis of the antioxidant tripeptide GSH, Ser, purines, creatine, and heme group
[24]. Evidence has suggested that rapidly growing cancer cells have a high Gly dependency
[99].
4.4. Arginine
Arginine (Arg) is an NEAA used for protein synthesis. It also participates in many other biological processes, including the synthesis of nitric oxide, creatinine, ornithine, agmatine, and polyamines
[24][100]. It also plays a key role in the urea cycle
[24]. Normal cells can synthesize Arg from citrulline and aspartate (Asp) through ASS1 (argininosuccinate synthase 1) and ASL (argininosuccinate lyase) in the urea cycle.
Arg-free diets can decrease the plasma levels of Arg in healthy volunteers. An Arg-free diet taken for 6 days reduced Arg plasma levels by approximately 20–40%
[101]. In another study, 4 weeks of a dietary restriction of Arg and other precursors of Arg (Asp, Pro, and Glu) significantly decreased Arg plasma levels without causing side effects
[102].
Mechanistically, dietary Arg deprivation may induce selective anticancer activity because many cancer cells express low levels of ASS1, which is involved in the synthesis of Arg. The downregulation of ASS1 facilitates cancer cell proliferation by increasing the aspartate availability for pyrimidine biosynthesis
[103].
The importance of Arg for cancer cell proliferation and survival has been supported by numerous studies that have shown that a pharmacological depletion of Arg levels with Arg-depleting enzymes induces anticancer activity. Two different enzymes are currently under clinical development: ADI-PEG20 (pegylated arginine deiminase) and PEG-BCT-100 (pegylated recombinant human arginase 1). These enzymes have shown anticancer activity in a wide variety of cancers, including melanoma, hepatocarcinoma, and glioblastoma
[100][104][105][106][107].
As occurs with other AAs, Arg restriction may have a negative impact on immunogenic cancers. Some cancer cells create an immunosuppressive microenvironment by converting myeloid cells into M2 macrophages or myeloid-derived suppressive cells
[108]. These immunosuppressive cells express arginase, which hydrolyzes Arg to ornithine and urea, therefore reducing the Arg levels in the tumor microenvironment
[108]. Arg is essential for T-cell proliferation and the expression of arginase can disrupt antitumor immunity
[108][109][110].
4.5. Glutamine
Gln is a non-essential proteinogenic AA that can be considered as essential under certain conditions
[111]. It is the most abundant AA in human plasma and tissues and is involved in many biological processes
[112]. It participates in the transport and detoxification of ammonia in the urea cycle, helping to maintain the pH balance
[113][114]. Gln is the main source of nitrogen atoms for the biosynthesis of nucleotides (pyrimidines and purines) and NEAAs (Glu, Asn, Ala, Asp, Ser, Pro, and citrulline)
[113]. Gln also mediates the cellular uptake of certain EAAs; for example, LAT1 imports the EAA Leu while simultaneously exporting Gln
[115].
Proliferating cancer cells have a high Gln demand. Cancer cells obtain high Gln levels by increasing their biosynthesis or by obtaining it from the extracellular environment
[113]. The increased Gln uptake of cancer cells has been associated with lower plasma levels of Gln in patients with several types of cancer
[116][117]. The increased Gln uptake by tumors is actually being studied for diagnostic purposes with PET imaging using 18F-(2S,4R)-4-fluoroglutamine
[118][119][120][121]. The increased Gln uptake of cancer cells is related to their high expression of ASCT2 (SLC1A5)
[122][123][124][125][126]; this chief Gln transporter is upregulated by the oncogenes MYC and KRAS
[127][128].
Limiting Gln levels and targeting Gln acquisition and utilization have been studied as possible anticancer strategies. Few studies have evaluated the in vivo anticancer activity of diets deficient in Gln. In 2017, the dietary restriction of Gln was found to induce anticancer activity in vitro and in vivo in a p73-expressing medulloblastoma xenograft model
[129]. The Gln-restricted diet increased mice survival and also showed a synergistic effect with cisplatin. Although the only difference between the control and experimental diets was the presence/restriction of Gln, both diets also lacked Glu, Ala, Asn, Asp, and Pro. This diet reduced the Gln and Glu levels in the cerebellum and cerebrospinal fluid of the mice
[129].
Most anticancer strategies targeting the altered Gln metabolism of cancer cells have focused on the pharmacological inhibition of Gln acquisition and utilization
[113]. These include the inhibition of GLS1 with inhibitors such as CB-839 (telaglenastat)
[130], BPTES
[131], and C.968
[132][133]. CB-839, which is orally bioavailable, has been tested in clinical trials. There are at least 21 completed or ongoing phase I-II clinical trials, 8 of which have been completed
[134]. In general, CB-839 was safe and well tolerated by cancer patients
[135][136][137][138][139][140][141]. In most of the completed clinical trials, CB-839 was combined with other anticancer drugs
[113]. Its benefit for cancer progression has been modest so far
[113]. There are other experimental anticancer drugs targeting Gln metabolism. The inhibition of Gln uptake by V-9302, an inhibitor of the ASCT2 transporter, induced anticancer activity in murine cancer models
[142]. JHU083, which is a prodrug of the Gln antagonist DON
[143], is selectively activated in the tumor microenvironment and disrupts cancer cell metabolism while improving T-cell anticancer responses. This compound induced marked anticancer activity alone and in combination with immunotherapies in several murine cancer models
[143][144][145][146][147][148].
4.6. Glutamate
Glu is an NEAA closely related to Gln. This AA is used in protein synthesis and has many other cellular functions. Glu is as a nitrogen donor for transaminases
[24]. It is used in the synthesis of many NEAAs, including Ala, Asp, Ser, Pro, and Gln
[35]. Transaminases and glutamic dehydrogenase (GDH) can convert Glu into αKG, which can be used to fuel the TCA cycle for energy production. In the brain, Glu is an excitatory neurotransmitter and can also be used for the synthesis of the inhibitory neurotransmitter γ-aminobutyric acid (GABA)
[24]. Glu participates in ROS protection by allowing CysS uptake by the xCT antiporter, and by directly participating in the synthesis of the tripeptide GSH
[149].
Although Glu supports cancer cell proliferation and survival, the anticancer activity of dietary Glu restriction has not been extensively studied, probably because Glu can be easily obtained from Gln, Asp, and Ala, and is also produced in the degradation pathways of many AAs, including Leu, Ile, Val, Lys, Phe, His, Tyr, and Pro.
4.7. Asparagine
Asn is an NEAA that can be synthesized from Asp by the enzyme ASNase. Asn is needed for protein synthesis, but the importance of Asn in other cellular processes is less understood
[150]. Asn can modulate mTORC1 activity and serve as an exchange molecule for the uptake of other AAs (e.g., Ser, Arg, and His), and the maintenance of intracellular Asn levels seems to be critical for cancer cell growth
[151].
Asn is commonly used to exemplify the relevance of NEAA restriction in cancer therapy, because the Asn-depleting enzyme ASNase is a useful drug for patients with acute lymphoblastic leukemia (ALL) and acute lymphoblastic lymphoma (ALLy). ASNase is an enzyme from
E. coli that deaminates Asn to Asp and ammonium; its intravenous administration quickly depletes the Asn from serum and cells
[152]. ALL cells usually rely on external Asn for their survival, and the depletion of Asn with ASNase leads to apoptosis in leukemia cells
[153]. ASNase is pegylated (PEG-ASNase) to extend its half-life and reduce the immunogenicity of the enzyme. Nowadays, ASNase is included in most chemotherapy regimens for pediatric ALL and ALLy, achieving high survival rates
[154]. The efficacy of ASNase is generally correlated with the expression of ASNS in leukemia cells
[155][156]; this enzyme allows the synthesis of Asn from Asp. However, in some cases, ASNS expression after ASNase has not been associated with resistance to treatment
[157]. ASNase also has Gln-depleting activity, which may participate in the anticancer activity of this enzyme
[158][159].
4.8. Aspartate
In addition to its role in protein synthesis, Asp participates in the synthesis of purines, pyrimidines, Asn, and Arg
[24]. It also plays a role in the urea cycle, the malate-Asp shuttle, and transamination reactions
[24]. Due to its role in the synthesis of nucleotides, Asp is crucial for proliferating cancer cells.
Although Asp can become a limiting factor for tumor growth, the antitumor activity of dietary Asp deprivation has not been evaluated individually, probably because this AA can be easily obtained from Glu and OAA through GOT1/2 (AST) transaminases (
Figure 1). Since these enzymes are expressed in many tissues, including the liver, a dietary Asp restriction would not result in a systemic Asp restriction.
4.9. Tyrosine
Tyr is an aromatic NEAA that can be obtained from the EAA Phe. In addition to its role in protein synthesis, Tyr is necessary for producing catecholamines (dopamine, epinephrine, and norepinephrine) and melanin
[24].
Since Phe is a precursor of Tyr, both AAs are usually restricted simultaneously in most cancer studies. A dual restriction of Phe and Tyr has been evaluated in animal studies and cancer patients with several positive results.
4.10. Alanine
Ala is a proteinogenic NEAA with other important metabolic functions. It is involved in transamination reactions and the glucose-alanine cycle (Cahill cycle). Ala can be easily converted into pyruvate by GPT1/2 transaminases
[24]; pyruvate is a carbon source for energy production, fatty acid biosynthesis, and gluconeogenesis
[24][160].
The antitumor activity of dietary Ala deprivation has not been evaluated independently of other AAs, probably because Ala can be easily obtained from Glu and pyruvate through GPT1/2 transaminases (
Figure 1). Since these enzymes are expressed in many tissues, including the liver, a dietary Ala restriction would not result in a systemic Ala restriction.
4.11. Proline
Pro is a proteinogenic NEAA that can be synthesized from Glu or ornithine
[161] (
Figure 1). Pro can be used for the synthesis of Arg, Glu, and polyamines, and participates in wound healing and the immune response
[24][162]. Like Gly, Pro is a major building block for the synthesis of collagen
[24]. Collagen is the main Pro storage in the human body
[161], and some cancer cells, such as pancreatic cancer cells, can use extracellular collagen to obtain Pro under conditions of nutrient deprivation
[163].
Dietary Pro restriction inhibited tumor growth in mice xenografted with PC-9 lung cancer cells, but not in mice with PaTu-8902 pancreatic cancer cells
[164]. Mechanistically, Pro starvation induced endoplasmic reticulum stress in cancer cells with a hyperactivation of mTORC1-mediated 4EBP1 signaling
[164].
5. Manipulation of Multiple Amino Acids Simultaneously
Since the metabolic routes of many AAs are interconnected (
Figure 2), the cellular requirements of specific AAs are probably influenced by the levels of other AAs. Manipulating several AAs simultaneously may therefore be more therapeutically useful than restricting AAs individually. As discussed in the previous sections, several pairs of AAs have usually been restricted together. Phe is the precursor of Tyr, and several studies have shown in vivo anticancer activity when both AAs were restricted together
[27][165][166][167][168]. Similarly, since Met can be used to synthesize Cys, a dietary restriction of both AAs has induced in vivo anticancer activity in different cancer types
[21][27][33][34][59][61][68][69][70][79]. The NEAAs Ser and Gly can be easily interconverted by SHMT1/2 enzymes (
Figure 1), and the simultaneous restriction of both AAs has induced anticancer activity in several murine cancer models
[96][97][169][170][171][172][173][174][175][176][177][178][179][180][181].
6. Conclusions
Cancer cells reprogram their metabolism to produce the large amounts of building blocks required for biosynthesis and proliferation, fulfill their high energy demands, and survive under conditions of elevated oxidative stress. The altered AA metabolism of cancer cells is one of most therapeutically relevant metabolic features of cancer.