Dietary restriction of amino acids for Cancer therapy
Biosyntheses of proteins, nucleotides and fatty acids, are essential for the malignant proliferation and survival of cancer cells. Cumulating research findings show that amino acid restrictions are potential strategies for cancer interventions. Meanwhile, ...
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Introduction
Cancer is a complex disease. There are more than 100 distinct types of cancer but sharing common hallmarks, including sustaining proliferative signaling and evading growth suppressors [1, 2]. The anabolic and catabolic metabolisms of cancer cells must be reprogrammed to maintain their proliferation and survival, and may even hijack normal cells to create tumor microenvironment (TME) for tumorigenesis and avoiding immune destruction [2]. Due to the demands of cell growth and the needs of newly synthesized proteins as direct effectors of cellular activities, protein biosynthesis is the most energy-demanding process that accounts for ~ 33% of total ATP consumption [3–5]. Interestingly, cumulating research findings have demonstrated that amino acid (AA) restrictions play roles in cancer interventions, including glycine restriction [6], serine starvation [7–9], leucine deprivation [10], glutamine blockade [11, 12], asparagine [13] and methionine [14]. These findings inspire and motivate a number of questions. Is there a common and effective metabolic intervention for cancer? For amino acids (AAs), which is the most heavily used AA in vivo? Which AA restriction is cell proliferation the most sensitive to? What kind of dietary strategies are practically available for cancer control?AA metabolism is the leading energy-consuming process
The consumption and release profiles of 219 metabolites gave us glimpses of the anabolic and catabolic features of the NCI-60 cancer cell lines [6]. The consumption profiles of cancer cells represented the homogeneous demands for energy metabolism and protein synthesis, which are vital biological processes for the malignant proliferation of cancer cells. The leading substrates consumed in cancer cells included glucose and AAs, such as tryptophan, tyrosine, phenylalanine, lysine, valine, methionine, serine, threonine, isoleucine, leucine and glutamine [6]. Meanwhile, the releases features of the NCI-60 cancer cell lines showed the nonhomogeneous catabolism of glycolysis and tricarboxylic acid (TCA) cycle [6].Another common feature of the NCI-60 cancer cell lines was the releases of nucleotides and nucleobases [6], which demonstrated that cancer cells did not directly consume nucleobases and nucleotides for their anabolism. RNA/DNA synthesis is the second energy-consuming process that contributes approximately 25% of total ATP consumption [3, 4]. It is well known that cells can use AAs (including asparate, glutamine, serine and glycine, Fig. 1a) as carbon and nitrogen resources for the syntheses of nucleobases [15–17]. Consequently, the AA metabolisms of cancer cells could use ~ 33–58% of the total energy expenditure (ATP) for protein synthesis and RNA/DNA synthesis (Fig. 1b). From the system point of view, the energy expenditure of AA metabolisms might sufficiently underline the important and potential roles of AA restrictions in cancer interventions.
Leucine is the most heavily used AA in human proteome
Since the consumptions and metabolisms of AAs are the most demanding biological processes for cancer cell growth, the most heavily used AA for protein synthesis might be a potential candidate for dietary restriction in cancer therapy. Thus, the percentages of 20 AAs for all proteins in human proteome (Uniport: UP000005640, Supplemental Table 1) were counted, sorted and plotted from the largest to the smallest percentages for comparison (Fig. 2a). As demonstrated, leucine is the most heavily used AA, serine is the second ranked, and tryptophan is the least used AA in the human proteome (Fig. 2).Leucine is an essential AA (EAA), so that it is somewhat out of expectation that leucine ranks first. Leucine is not only necessary for protein synthesis, and also acts as a signaling molecule activating mechanistic target of rapamycin (mTOR) signaling through sestrin-2 that is a cytosolic leucine sensor [18–21]. Interestingly, EAAs may ideally act as a key signal for amino acid availability. Specifically, leucine is one of branched chain AAs (BCAAs), which are not first catabolized in the liver due to the low activity of BCAA aminotransferase [22]. Consequently, leucine increases rapidly in circulation after meal [22], and are readily available as an essential nutritional signal to reduce food intake via mTOR-dependent inhibition of hypothalamic Agouti-related protein (Agrp) gene expression [23, 24].
Although leucine is the highest enrichment AA in proteins, leucine deprivation showed modest effects on human breast cancer cells [10]. The exceptional abundance of leucine in human proteome might partially explain its limited effects on cell proliferation since leucine in degraded proteins may efflux from lysosome to meet the demand of growth [25].
Serine is the second frequently used AA
Serine is a non-essential AA (NEAA) and ranks second. Serine metabolism is altered and enhanced in cancer cells [26–28]. The enhanced serine synthesis pathway could make significant contribution (~ 50%) to the anaplerosis of glutamine to α-ketoglutarate for mitochondrial TCA cycle [26]. Serine starvation induced stress and promoted p53-independent and p53-dependent metabolic remodelling in cancer cells [7]. Under the starvation of serine, the upregulation or enhancement of de novo serine synthesis pathway and oxidative phosphorylation were independent of p53, while the inhibition of nucleotide synthesis was dependent of p53-p21 activation so that the limited amount of de novo serine was shunted to glutathione production for the survival of cancer cells [7]. Therefore, serine starvation might have a potential role in the treatment of p53-deficient tumors.Glycine restriction and supplement
Although glycine might be important for rapid cancer cell proliferation by supporting de novo purine nucleotide biosynthesis [6], glycine restriction alone didn’t have the same detrimental effect on cancer cells as serine starvation, which might be explained by the inter-conversion between serine and glycine in one-carbon metabolism by serine hydroxymethyl transferase (SHMT) [7, 16, 17], especially the mitochondrial glycine synthesis enzyme SHMT2 [6]. Interestingly, the consumption and release profiles of the NCI-60 cancer cell lines demonstrated that glycine among AAs had the most heterogeneous pattern either consumed or released, whereas serine showed relatively homogenous consumption [6]. Beyond the potential role of glycine restriction in blocking the rapid growth of certain cancer cells, the dietary supplement of glycine was also reported to inhibit the growth of certain types of tumors, such as liver tumors [29] and melanoma tumors [30]. Therefore, the heterogeneous metabolism of glycine in cancer cells might account for its paradoxical effects.Lysine is a particularly important EAA
NEAA restrictions play limited roles in cancer therapy since there are de novo synthesis pathways, such as serine [7]. Therefore, EAAs were focused and plotted with a log scale so that we could have a close view of EAA enrichment profiles (Fig. 2b). Lysine is particularly noteworthy (Fig. 2b). The AA compositions (represented in percentages) of 14 plants and nine animals were analyzed, and the AA medians and standard deviations (SD) of these proteomes were plotted and compared with human AA abundances (represented as the width of bubbles in Fig. 2c, Supplemental Table 2 and 3). The relative abundances of AAs in plants, animals or human were almost identical with subtle differences, and lysine ranked in the middle upper level of AAs (Fig. 2c).Those EAAs rich proteins might be particularly vulnerable to EAA restrictions, so that the 3-sigma upper limit (median + 3x SD) was applied to sort EAA exceptional rich proteins (ERPs). The numbers of ERPs rang from 604 to 1298 for individual EAA (Supplemental Table 4). The averaged percentages (in median ± SD) of lysine, valine and leucine in human proteome were 5.26% ± 3.27, 5.88% ± 2.50 and 9.9% ± 3.71% respectively (Fig. 2a). In comparison, the averaged percentages (in median ± SD) of lysine ERPs (n = 918), valine (n = 595) and leucine ERPs (n = 604) were 16.99% ± 3.65, 14.89% ± 2.62 and 23.28% ± 4.5% respectively (Fig. 2b). Interestingly, although the abundance of lysine in human proteome ranked third after leucine and valine (Fig. 2a and c), the abundance of lysine ERPs ranked second only after leucine (Fig. 2b). The lowest averaged percentage of ERPs was tryptophan ERPs (6.05% ± 1.86%, n = 1298) (Fig. 2b).
ERPs were further chosen for functional enrichment analysis with the web server of g:Profiler [31]. To our surprise, none term of gene ontology – molecular function (GO-MF) was met the significant threshold (p < 0.001) for leucine, whereas lysine ERPs showed a number of enriched GO-MF terms including DNA/RNA/chromatin binding, structural constituent of ribosome, nucleosome binding and so on (Fig. 2d). Together, these suggested that lysine and its ERPs were very important for cell functions, and that cell growth might be particularly vulnerable for lysine restriction. Indeed, lysine deprivation could completely block the proliferation of either p53-competent or p53-deficient cancer cells [7].
Importantly, kwashiorkor is a severe protein malnutrition disease of childhood associated with lysine deficiency in normal maize diet [32]. Normal maize has more protein than rice but containing low levels of two EAAs - lysine and tryptophan, which lead to the imbalance of amino acids and malnutrition [32, 33]. Nowadays, maize as one of daily staples is biofortified and named as quality protein maize (QPM). QPM contains an opaque-2 gene. The opaque-2 gene codes a transcriptional activator so that QPM expresses more lysine and tryptophan rich proteins [33]. In the Williams’ report about kwashiorkor first published in 1933 and republished in 1983, five cases were described in detail; all cases had a history of lacking breast-feeding, and were only fed with the food prepared from normal maize (cassava also used in case 5); it took 4 to 12 months for the development of the kwashiorkor disease in those children [32]. According to the abundance of lysine and tryptophan as shown in Fig. 2, lysine deficiency might be the leading cause for kwashiorkor since the averaged percentage of tryptophan in proteins was 1.17% ± 1.3% (median ± SD) and significantly less than the abundances of other AAs (Fig. 2).
Besides the important functions of lysine ERPs and the growth-halting effects of lysine deprivation discussed above, lysine is also a versatile AA modified by various modifications including methylation, acetylation, phosphorylation, malonylation, O-GlcNAcylation, SUMOylation, ubiquitination and lactoylation, especially those lysine residues in histones [34–36]. These post-translational modifications of lysine and its ERPs regulate the structures and functions of enzymes to expand the functional proteome [36], represent the crosstalk between metabolism and epignome [37], and also link cell signaling and metabolic reconfiguration to cell proliferation and differentiation [38]. Thus, all these support that lysine is an important EAA, its ERPs and their modifications play indispensable roles in homeostasis, proliferation, differentiation and diseases including malnutrition and cancer. However, the research or clinical data about lysine restriction on cancer intervention are very limited so far, and deserve more elegant efforts.
Tryptophan is the least used and available EAA
Tryptophan is an interesting and unique AA, which is the least used (Fig. 2a) and also the least available AA from animal or plant foods (Fig. 2c). As an EAA, tryptophan cannot be synthesized in vivo, and must be acquired from foods. This particular characteristic of tryptophan gave its additional roles beyond as a necessity in protein synthesis. For instance, immune system could induce tryptophan degradation to inhibit the growth of certain cancer cells [39] via interferon gamma (IFN-γ) upregulating the tryptophan-catabolizing activity of indoleamine 2,3-dioxygenase (IDO) [40]. Meanwhile, cancer cells could use the same mechanism to impede and escape immune response in TME [41, 42]. Consequently, the clinical trials of IDO inhibitors showed limitations and off-target effects due to the multifaceted tryptophan metabolism [43].The metabolism of tryptophan might be the most complicated one among AAs, and was involved in the regulation of immunity, neuronal function and intestinal homeostasis [44]. Majority (~ 95%) of absorbed tryptophan degraded via kynurenine pathway, in which IDOs and tryptophan-2,3-dioxygenase (TDO) were the main rate-limiting enzymes. Besides the usage for protein synthesis, a small fraction of tryptophan was catabolized by tryptophan hydroxylase (TPH) for the production of serotonin (5-hydroxytryptophan, 5-HT) and melatonin. Tryptophan and its metabolites were used and catabolized by various organs and cells to further generate bioactive metabolites, including neuroprotective kynurenic acid by astrocytes, neurotoxic quinolinic acid by microglia, neuromodulator tryptamine, immune suppressive metabolites (such as 3-hydroxykynurenine, 3-hydroxyanthranilic acid and xanthurenic acid) [42, 44]. The catabolism of tryptophan induced by IFN-γ in cancer cells and macrophages showed that the catabolites of tryptophan differed in kynurenine, anthranilic acid and 3-hydroxyanthranilic acid [40].
Tryptophan was mainly catabolized by TDO in liver, then oxidized to acetoacetyl-CoA and used for the synthesis of nicotinamide adenine dinucleotide (NAD+) [44]. Thus, TDO knockout mice showed increased levels of tryptophan in plasma and 5-HT in the hippocampus and midbrain, and hence demonstrated anxiolytic modulation and adult neurogenesis [45], which was consistent to the cumulated evidence about the role of 5-HT in neurogenesis and anti-depression recently intensively reviewed by us [46, 47]. It was also well known decades ago that 5-HT was involved in food intake and mood [48], and recent research focused on the functional modulation of 5-HT6 receptor including its agonists and antagonists [49, 50]. Both the agonists and antagonists of 5-HT6 receptor could reduce food intake [49, 50], which suggested that the activation curve of 5-HT6 receptor was likely a bell shape and that a proper concentration of 5-HT might increase food intake through 5-HT6 receptor. In short, tryptophan metabolism and its catabolites played active roles in proliferation, immunity, neurogenesis, anxiety, depression and food intake under physiological conditions.
Methionine is the penultimate EAA
Methionine is essential for the initiation of protein synthesis, while N-formylmethionine-tRNA generated in mitochondrial folate cycle is the initiator of mitochondrial protein synthesis [51] (Fig. 3). SAM provides the methyl group for epigenetic modification. The methionine cycle coupling with mitochondrial energy metabolism produces SAM, so that SAMTOR might sense not only methionine, also one-carbon resource and energy levels. The methionine cycle and folate cycle are two functional modules connected and involved in one-carbon metabolism [16, 17, 52]. Serine acts as one-carbon donor and tetrahydrofolate (THF) serve as one-carbon receptor in the folate cycle [17]. In the methionine synthase reaction (vitamin B12 as a essential cofactor), methyl-THF donates its methyl group via vitamin B12 to homocysteine to produce methionine and THF [53]. The one-carbon metabolism of cancer cell may mobilize multiple carbon sources including glucose, serine, threonine, glycine, formate, histidine and choline [52]. The syntheses of pyrimidine and purine nucleotides require carbon and nitrogen sources, which rely on AA metabolism and the folate cycle in accordance with the energy metabolism and activities of mitochondria [15]. In the folate cycle, THF is an essential factor for nucleotide synthesis and the survival of cancer cells, and formyl-THF serves as one-carbon reserve [52]. Clearly, one-carbon metabolism is essential for multiple physiological processes (Fig. 3) including nucleotide metabolism (especially purine synthesis), glutathione (synthesized from glutamate, cysteine and glycine) and NAPDH synthesis for antioxidant defense [15, 52]. Consequently, one-carbon metabolism and nucleotide metabolism were altered and involved in the effects of the restrictions of glycine, serine and methionine [6, 7, 14]. Interestingly, the dietary supplementation of histidine upregulated the histidine degradation pathway to deplete THF and then enhanced the sensitivity of cancer cells to methotrexate, an inhibitor of dihydrofolate reductase for THF synthesis [54].[...]
Summary
Now, we can systematically answer the four questions raised at the beginning. First, AA restrictions could be a common and effective metabolic intervention for cancer since the AA metabolisms could use up to 58% of the total ATP for protein synthesis and RNA/DNA synthesis (Fig. 1b). Second, leucine is the most heavily used AA in human proteome; serine ranks the second; tryptophan is the least used and available EAA. Third, lysine is a particularly important EAA as discussed above, which restriction the proliferation of cancer cells might be most vulnerable and sensitive to. Fourth, it is recommended that the most practical dietary strategy for cancer intervention is using normal maize as an intermittent staple food for days, weeks or even months for lysine restriction, and starchy foods, vegetables and fruit serving as complementary foods to meet daily micronutrient needs and for a rich and varied diet.This retrospection and perspective focused on AA restrictions in cancer interventions. Consequently, few information for many other AAs was available to be included and summarized here. On the other hand, some AA supplements were discussed, such as glycine [29, 30] and histidine [54]. The glycine supplement could inhibit the growth of liver tumors [29] and melanoma tumors [30]. The histidine supplement upregulated the histidine degradation pathway and then enhanced the sensitivity of cancer cells to methotrexate chemotherapy [54]. Arginine, glutamine and cysteine were mentioned as immunonutrition [99]. Arginine supplement affecting thymus (one of primary lymphoid organs), could significantly increase thymic weight (~ 22%) and thymic lymphocyte content (~ 45%) [110], which was consistent to its role as an important signaling molecule for growth and immunonutrition. Tryptophan as the least used and available EAA, was very unique so that both cancer cells and immune cells used its catabolism to inhibit the growth of opponents. Thus, the supplementation of tryptophan and arginine was further recommended to enhance the proliferation and function of T cells.
As for AA restrictions in cancer interventions, cumulating research findings of AA restrictions had been discussed, including glycine restriction [6], serine starvation [7–9], leucine deprivation [10], glutamine blockade [11, 12], asparagine [13] and methionine [14]. NEAA restrictions played limited roles in cancer therapy since there were de novo synthesis pathways, such as glycine [6] and serine [7]. Glycine restriction was only effective for rapid cancer cell proliferation, while serine starvation was only fitted to the treatment of p53-deficient tumors [6, 7]. Leucine deprivation showed mild effects on human breast cancer cells [10], which might due to its exceptional abundance in human proteome. A new derivative of DON, JHU083, blocked glutamine metabolism in TME and induced a metabolic reprogramming of effector T cells relied on acetate to overcome tumor immune evasion [12]. Asparagine restriction could reduce the metastasis of breast cancer without affecting the growth of the primary tumour [13]. Methionine restriction alone demonstrated mild effects on the proliferation of cancer cells, but enhanced the sensitivity of cancer cells to chemotherapy and radiation [14]. About the recommended strategy of lysine restriction, there was little information and study to support or verify its potentials in cancer therapy by this time, so that works about the effects of lysine restriction on various cancer types were expected to be flourishing in near future.
