Asparagine: The Underdog of Cancer Cell Growth

It is challenging to come across current scientific literature regarding cancer research without running into the involvement of glutamine. The evidence that tumor cells exhibit high rates of glutamine consumption and the role of glutamine in the synthesis of lipids, nucleotides, and amino acids is known. Regardless, glutamine remains a hot topic in cancer research. As we briefed upon in class, glutamine adds carbons to the tricarboxylic acid cycle (TCA cycle) through a process known as anaplerosis in order to replenish TCA cycle intermediates that have been extracted for biosynthesis1. Although glutamine is able to participate in the synthesis of several amino acids through its catabolism to glutamate, asparagine is the only amino acid that requires glutamine for de novo synthesis. Glutamine is a substrate for asparagine synthetase (ASNS), which converts aspartate to asparagine. The reaction is ATP-dependent and unidirectional, which suggests that the production of asparagine occurs at the expense of biosynthesis production, since glutamine is shifted away from anaplerosis2.

The structural similarity between glutamine and asparagine (the two only differ by one carbon on the functional amino acid side chain), as well as the dependence on glutamine for asparagine synthesis suggests that asparagine may also play a similar role to glutamine in amino acid regulation. For example, ASNS expression is upregulated when amino acid concentrations are limited. So, what effect does an increase in asparagine have on the cell? The only currently known direct impact of asparagine on the cell is that it acts as a substrate for protein synthesis. In a recent 2016 issue of Nature Communications, Krall et al2uncovers a novel role for asparagine in amino acid homeostasis.

Due to the known contribution of glutamine on cancer growth, drug therapies have already been developed targeting glutamine metabolism in the form of glutaminase (GLS) inhibitors, which inhibit the formation of glutamine from glutamate. The authors hypothesized that glutamine–dependent cancers may generate resistance to GLS inhibitors by relying on metabolites downstream of glutamine. In particular, the authors were interested the relationship between glutamine and asparagine levels on cell. To test this, the authors generated sarcoma cells that grew in the absence of glutamine (glutamine-independent cells), as well as breast cancer cells that were resistant to GLS inhibitors. Interestingly, both cell types experienced a decrease in cell proliferation when exogenous asparagine was removed, even when glutamine levels were unchanged. This was shocking since glutamine-independent cells presumably synthesize sufficient concentrations of glutamine for protein and nucleotide synthesis. However, the lack of cell growth suggested that asparagine was needed for cell proliferation, even if glutamine levels were sufficient.

The prominent role of asparagine in cell proliferation was further confirmed by knocking out ASNS in cancer cell lines in glutamine-abundant conditions. Cells with the dysfunctional enzyme form exhibited a reduced proliferation rate, which then was recovered to control levels with supplementation of an asparagine-containing medium. Thus, intracellular production of asparagine impacted cancer cell proliferation even in glutamine-abundant conditions.

Next, the authors sought to investigate the mechanism to how asparagine enables cancer cells to proliferate. Previous research has demonstrated that intracellular glutamine has the capacity to swap with extracellular amino acids via a transport protein. Given the structural similarity between glutamine and asparagine, as well as an undetectable net influx of asparagine, it was hypothesized that asparagine was being imported into cancer cells to only be exported in exchange for other amino acids. The potential role of asparagine facilitating the transport of amino acids was determined by examining the change of extracellular asparagine concentrations in the presence of an amino acid medium that did not contain asparagine. After treatment with the amino acid-abundant medium, asparagine was detected in the medium and intracellular asparagine levels were depleted. The increase of extracellular asparagine levels and the decrease in intracellular asparagine levels demonstrated that asparagine allowed for amino acids to enter the cell at the expense of asparagine leaving the cell. In particular, it was determined that asparagine preferentially exchanges with serine, threonine, and non-polar amino acids over basic amino acids. This phenomenon was experienced even in cells that were asparagine deficient.

Furthermore, the authors reasoned that if both glutamine and asparagine function as amino acid exchange factors, is there a cellular preference towards one amino acid over the other? When both asparagine and glutamine intracellular concentrations were high and the cancer cells were exposed to an amino acid containing medium, only the efflux of asparagine was detected, suggesting that the use of asparagine as an amino acid exchange factor may be preferable over glutamine. Furthermore, when inhibiting asparagine synthesis by knocking out ASNS, amino acid influx was impaired, but was then relieved with supplementation of exogenous asparagine. These results indicated that amino acid exchange with asparagine was favored over glutamine. Asparagine did not simply act as a substitutive for glutamine under glutamine-depleted conditions.

Thus, it was logical to predict the exchange factor role of asparagine would influence protein synthesis in cancer cells. In particular, it is known that mammalian target of rapamycin complex 1, (mTORC1) activity is induced by the presence of intracellular amino acids. Stimulation of mTORC1 activates downstream translation factors through the phosphorylation of eukaryotic initiation factor 4E (eIFAE)-binding protein, which permits the assembly of the translation ignition complex (Figure 1). ASNS knockout caused an increase of the inhibitory form of eIFAE-BP, preventing the translation complex form forming. Thus, asparagine’s role in amino acid exchange enabled intracellular protein synthesis.

In a similar manner, it was determined that asparagine also has an impact on nucleotide synthesis. In addition to the prior mechanism, mTORC1 activation also promotes nucleotide synthesis by increasing phosphorylation of CAD (carbomoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase), which catalyzes the first three steps of de novo nucleotide synthesis (Figure 1). ASNS knockdown lowered mTORC1 activation and intake, suggesting that asparagine also played a role in nucleotide synthesis.

Through this study, the authors uncovered a previously unrecognized role for asparagine as an amino acid exchange factor, as well as having roles in both protein and nucleotide synthesis. Therefore, both glutamine and asparagine are major contributors to cancer cell growth. Undoubtedly, this can have clinical implications for targeting asparagine metabolism as cancer therapies. The authors suggested that coupling asparagine inhibition treatment with a low glutamine diet or coupling glutamine inhibition treatment with an asparagine-low diet would be effective cancer treatment by targeting both asparagine and glutamine metabolism. Although the data suggested that asparagine exchanges with extracellular amino acids, it still remain unclear which membrane transporters are involved in this process. Future research can be used to identify the specific transporters involved, which may also be effective drug targets for the inhibition of cancer growth,

Figure 1: The cellular impact of amino acid influx on protein synthesis and nucleotide synthesis. Intracellular asparagine exchanges with extracellular amino acids (AA), such as serine, which is known to have an effect on nucleotide synthesis.

Nature Article: Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor

References:

1. Hosios, A. M. et al. Amino Acids Rather than Glucose Account for the Majority of Cell Mass in Proliferating Mammalian Cells. Developmental Cell 36, 540–549 (2016).

2. Krall, A. et al.. Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor. Nature Communications 7, 11457 (2016).

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