Nitrogen is a building block of all proteins in the body, RNA and DNA, so tumors are greedy for this element. When the body uses nitrogen, it generates a nitrogenous waste substance called urea from leftovers in a chain of biochemical reactions that occur in the liver, which are known as the urea cycle. As a result of this cycle, urea is excreted into the blood and is subsequently excreted from the body in the urine. Some nitrogenous compounds can derive from urea to build the building blocks for higher macromolecules such as nucleic acids. And it may seem strange, urea can do it: Not as such, of course, but through the urea cycle it can be converted to carbamyl groups that will serve the initial enzymatic reaction for the production of pyrimidines, one of its types. of DNA bases. A few years ago, a joint team of researchers from the Weizmann Institute of Science, the National Cancer Institute (NCI / NIH) and other institutes demonstrated that in many cancers, the patient’s nitrogen metabolism is altered, producing changes detectable in body fluids and contributing to the emergence of new mutations in cancerous tissue.
In previous research, the Weizmann Department of Biological Regulation team showed that one of the urea cycle enzymes was inactivated in many cancerous tumors, increasing the availability of nitrogen for the synthesis of pyrimidines (cytosine and thymine), which support cancerous growth as constituents of nucleic acids. Nucleotide synthesis is a key event for maximal cell proliferation due to a limited amount of intracellular nucleotide pools. Therefore, the enzymes involved in the nucleotide biosynthetic pathway are attractive targets for the growth inhibition of malignant cells. Nucleotide biosynthesis uses ribose 5-phosphate, produced by the oxidative and non-oxidative arms of the pentose phosphate pathway, and non-essential amino acids. The rate limiting step in this pyrimidine synthesis pathway is catalyzed by the carbamyl phosphate synthetase II (CPS 2) of the CAD enzyme complex. CAD activity is regulated by two molecules: phosphoribosyl-pyrophosphate (PRPP), synthesized from ribose 5-phosphate, increases the CPSase activity of CAD, while UTP (the final product of the synthesis) negatively regulates the CPSase activity by feedback inhibition.
Phosphorylation of CAD by mitogenic protein kinases (MAPK) and PKCα modifies the sensitivity of CAD to UTP and / or PRPP to regulate pyrimidine synthesis. Recently, CAD has been shown to be phosphorylated on residue 1859 by S6K and this phosphorylation stimulates CAD activity. S6K or Rsk-1 is a downstream kinase that MAPKs use to trigger protein synthesis. Another cellular effector activates CAD, the small G Rheb protein. It is a member of the Ras small GTPase superfamily which plays an important role in regulating protein synthesis, growth in response to nutrients and growth factors, and interacts with the mTORC1 complex. It seems that it collaborates with sestrin-1, which researchers have very recently identified as the potential activator of the mTORC1 complex in response to cellular load with branched-chain amino acids (leucine, isoleucine, etc.). Cancer cells do not waste anything, nitrogenous substances are essential for their replication. The most classic example of an amino acid that displaces nitrogen groups and which cancer cells are greedy for is glutamine, a non-essential but multifunctional amino acid.
In the study, conducted by the NCI and Weizmann, the team identified a number of precisely defined alterations in further enzymes of the urea cycle, which together increase the availability of nitrogen intermediates for pyrimidine synthesis. These alterations lead to increased levels of pyrimidine in the tumor and predispose the cancer to mutations. This seems a contradiction, given that the greater availability of pyrimidines can not only serve the synthesis of nucleic acids, but also have a spare pool for the possible repair of DNA damage. When the researchers made changes in the expression of urea cycle enzymes within colon tumors in mice, these mice – unlike the control group – had lower blood urea levels in addition to detectable levels of pyrimidine. in the urine. Next, the researchers examined the medical records of 100 pediatric cancer patients treated at Tel Aviv’s Sourasky Medical Center to check urea levels. At the time of admission, almost all the children had much lower urea levels than normal, a sign that the tumor (whatever type they had) was picking up nitrogenous waste to convert them back.
Furthermore, after examining large sets of genomic cancer data, the researchers found that urea cycle dysregulation is prevalent in many cancers and is accompanied by specific mutations resulting from increased pyrimidine synthesis. These pyrimidine-related mutations are a double-edged sword. On the one hand, they make the cancer more aggressive, reducing patient survival, but they also generate protein fragments that make the tumor more “sensitive” than average to the impact of the immune system. Therefore, tumors with a dysregulated urea cycle are more likely to be susceptible to immunotherapy, in which the patient’s immune mechanisms are directed to fight the tumor. An analysis of patients with melanoma revealed that those with tumors with dysregulated urea cycle enzymes were more likely to respond to immunotherapy than those without these characteristics. When the researchers induced dysregulation of urea cycle enzymes in the tumors of the mice, they found that the mice responded much better to immunotherapy than those with tumors with intact activity of the same enzymes.
Dysregulation in urea enzyme expression levels in tumor tissue would suggest that the patient is more likely to respond to immunotherapy. Another possibility the researchers think is worth exploring is whether genetic manipulation of the tumor to induce such dysregulation before immunotherapy could increase the effectiveness of the therapy. This manipulation would deliberately result in the destruction of the tumor’s urea cycle in the hope that this disorder will generate mutations in pyrimidine-bound proteins, helping the immune system to identify and destroy the tumor. Not only that, in the future, predicting the responsiveness of the tumor to immunotherapy could simply be deduced through the specific histological marking for the enzymes of the urea cycle. And not only for leukemia or melanoma, but for other types of cancer with a high rate of cellular duplication and aggressiveness, such as breast, pancreatic and lung cancer. Precisely in lung cancer called adenocarcinoma, when it depends on mutations of the LKB1 gene, the CPSase 1 enzymatic activity is central to the regulation of its nitrogen metabolism.
Carbamyl phosphate synthase 1 (CPS 1) is the first rate-limiting mitochondrial enzyme in the urea cycle. It must have a cross point with pyrimidine metabolism initiated by CPS 2, because if lung adenocarcinoma cells taken from biopsy specimens are treated to repress the CPS 1 gene, they suffer a severe proliferative arrest stroke. This is demonstrated by the lower overall rate of synthesis of DNA and nucleotide precursors (especially UTP and ATP). Indeed, the tumors with the highest CPS 1 expression were collected from patients with the worst clinical prognosis. The same has been seen for malignant liver tumors (hepatocarcinomas). Often these tumors show a consistent loss of function of the ASS1 gene, which codes for arginosuccinate synthetase, one of the final enzymes of the urea cycle. When liver tumors are treated in combination with arginine and 5-fluorouracil deprivation, the potency of this chemotherapy increases dramatically. In fact, by removing arginine from nitrogen metabolism, cancer cells not only do not have the amount of amino acid necessary to incorporate it into cellular proteins, but not even that which will be used by the urea cycle to connect with pyrimidine synthesis.
Thus, the drug will have all the power to block thymidylate synthetase, the last enzyme in the synthesis of pyrimidines. And this is also a possibility for colon and colorectal cancer, whose established chemotherapy cornerstone has been 5-fluorouracil and its derivatives. These types of cancer are among the most frequent in oncology and often have a poor prognosis if not diagnosed in time. Often this also depends on the degree of cellular differentiation and aggressiveness. A few years ago, researchers discovered that colorectal cancers may also be susceptible to arginine deprivation, but it all depends on whether they show deficiencies in the enzymes of the urea cycle. If they are deficient in the ASS1 and / or OTC enzymes for the urea cycle, then they may be susceptible to arginine-depriving therapy. As with liver tumors, also in those of the colon the depletion of arginine is obtained via enzymes. Arginine deiminase (ADI) conjugated with a molecular “tag” is administered which prolongs its half-life and prevents internal degradation (ADI-PEG20). But if the upstream root is the CPS 1 enzyme, why aren’t its inhibitors used? The answer is simple, there are no safe and low-toxic ones available.
A compound potentially capable of inhibiting CPS 1 and 2 (without discriminating) is alpha-amino-oxyacetic acid or 1-hydroxy-glycine which has never found uses outside the study of cellular metabolism in the laboratory. Beside, researchers have generally focused their interest on purine synthesis and indeed inhibitors of the purine early steps (fosfinothricin, AICAR) have been synthesized but virtually no early step inhibitor for pyrimidine pathway does exist. But last year, a team of researchers from H3 Biomedicine Inc. discovered a class of inhibitors called “allosteric” for the CPS 1 enzyme. They are derivatives of dimethyl-piperazine and act in a protein pocket other than the catalytic site. The good news is that they appear to bind this molecular “pocket” of CPS 1 without affecting the CPS 2 counterpart. These newly discovered molecules are a first step for researchers to have a tool for probing CPS1 in cancer biology. Given that nitrogen pathways are seemingly dysregulated in several types of cancers, this could be the long-seeked chance to hit many targets with just one bullet.
- Edited by Dr. Gianfrancesco Cormaci, PhD, specialist in Clinical Biochemistry.
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