Author: Brandon Copping
This figure shows the glutaminolysis pathway and the predicted mechanism for how cancer cells are able to reincorporate ammonia into producing building blocks for proliferation.
Cancer is, and has been since its discovery, a very complex and often very mysterious disease. For an affliction that takes the lives of millions across the world every year, much is still unknown about even what would seem to be the simplest of processes.1 One of these processes that we are still discovering mechanisms and pathways in is how cancer cells handle toxic byproducts of metabolic reactions. Understanding how cancer cells handle these toxins could provide us with useful insight on ways in which we could cripple cancer cells by impairing their methods of detoxifying these poisonous compounds that are produced. A recent paper by Spinelli et al. published in late 2017 in Science took a look at how cancer cells handle disposal, or lack thereof, of one of the simplest toxic byproducts: ammonia.2 What really stands out about this study is that, in healthy cells, ammonia is sent to the liver to be used in the urea cycle and thus detoxified and excreted. However, as this paper shows, that is not the case in cancerous cells. It has been shown before that metabolites typically thought of as poisonous to cells tend to accumulate in and around tumors, leading researchers to hypothesize that cancer cells must have some way of handling these toxins that is separate from the body’s typical response to detoxify and excrete.3 Spinelli et al. note that though much research has been done into lactate usage in cancer, little has been done to determine what is done with the excess ammonia.
In cancer cells, the typical source of ammonia is from amino acid metabolism, specifically glutamine and asparagine metabolism. What the authors wanted to find out is how this free ammonia could possibly be used by the cancer cell in some way that allows it to proliferate more effectively. In order to determine this, the authors first investigated the expression of three enzymes they knew were capable over overcoming the thermodynamic challenge of reattaching ammonia to a carbon skeleton: carbamoyl phosphate synthetase I (CPS1), which is the rate-limiting step of the urea cycle; glutamate dehydrogenase (GDH), an NADPH-dependent enzyme that aminates α-ketoglutarate; and glutamine synthetase (GS), which aminates glutamate into glutamine. After looking at levels of expression of each of the enzymes across various types of cancer cells, they determined that only GS and GDH expression was significantly increased in a majority of cancers. For their experiment they decided to use estrogen receptor-positive (ER+) breast cancer cells since they had even higher expression of GS and GDH when compared to other cancers.
To determine where the nitrogen in the ammonia was ending up in the cancer cells, they supplied the cell with glutamine that had the nitrogen-14 of the amide group swapped for nitrogen-15, a technique that allows for nitrogen tracing through mass spectrometry of metabolic derivatives. As they expected based on previous literature, 15N was found in asparagine as well as in nucleotides synthesized by the cell. However, there were a number of metabolites that contained 15N that they did not expect to: proline, aspartate, branched-chain amino acids, and glutamate. This result was significant because there was no known connection between the amide nitrogen of glutamine and any of these products. The presence of 15N in these molecules led the authors to believe that the enzyme of interest in this reaction is GDH, due to it producing glutamate which is upstream of the metabolites they were not expecting to be labeled.
In order to know for certain that it was GDH catalyzing the reincorporation of ammonia, the authors knocked out GDH in the breast cancer cells and then exposed them to the same labeled glutamine as before. What they found is that the incorporation of the labeled nitrogen into the unexpected metabolites significantly decreased. This result strongly suggests that it is, in fact, GDH that is reincorporating the freed ammonia. However, all of the freed, labeled ammonia up to this point had been liberated from the labeled glutamine via glutaminolysis. What the authors wanted to determine next was if GDH could incorporate any free ammonia in the cell, not just ammonia liberated from glutaminolysis. Since GDH is directly involved in glutaminolysis, it would consistently be in close proximity to the free ammonia released by this process. To see if this was possible, the authors treated the breast cancer cells with concentrations of 15N-labeled ammonium chloride at various concentrations. One of the surprising results they found was that even at concentrations of 10 mM (healthy concentration of ammonia ranges from 0-50 μM) the cancer cells showed next to no indication that there was any negative effect on them. In terms of the fate of the ammonia, however, they discovered that up to 20% of the glutamate in the cancer cells contained labeled nitrogen, suggesting that incorporation of freed ammonia is a very important process in cancerous cells.
Lastly, the authors wanted to determine whether these high concentrations of ammonia were beneficial to cancer cell proliferation or whether they were more neutral. To do this they cultured cancer cells and either swapped out the medium every day, thus not allowing much ammonia to accumulate, or every three days, allowing the ammonia to accumulate. What they found was that, when ammonia was allowed to accumulate, the proliferation rate of the cancer cells increased significantly. In order to confirm that all of the aforementioned processes, which were tested in vitro, were also taking place in vivo, they injected labeled ammonia into mice that had been infected with breast cancer cells. As expected, the mice that were injected with ammonia experienced increased tumor growth when compared to infected mice that were not injected with ammonia. Additionally, the in vivo incorporation of the labeled ammonia matched that which they found in vitro.
Since tumors are usually very poorly vascularized, it would stand to reason that the cancer cells would prioritize recycling ammonia in order to meet the high demand for nitrogen in the cell. A proliferating cell has a higher demand for building blocks, such as amino acids and nucleotides, than a differentiated cell does, so the cancer cells developed a process by which they can recycle ammonia in order to continue proliferating efficiently. Armed with this knowledge, it seems highly possible that this pathway may be able to be targeted by pharmaceuticals in order to neutralize the cancer cells’ ability to handle the toxicity of ammonia. Given the poor vascularization of tumors, knocking such a significant source of nitrogen could be a very useful way to stifle proliferation and slow, stop, or possibly even reverse tumor growth.
Link to paper: http://science.sciencemag.org/content/early/2017/10/11/science.aam9305.full
- Spinelli, et al. Metabolic recycling of ammonia via glutamate dehydrogenase supports breast cancer biomass. Science 358, 941-946 (2017).
- Eng, et al. Ammonia derived from glutaminolysis is a diffusible regulator of autophagy. Science Signaling 3, ra31 (2010).
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