The first portion of the unit we are currently studying revolved around the metabolism of nucleic acids. The nucleobases that serve as the building blocks of these molecules can be broken down into two categories, the dual-ringed purines, and the single-ringed pyrimidines. Purines consist of the bases Adenine, Guanine, and Inosine. Biosynthesis of the two genetically relevant bases, Adenine and Guanine occurs first via the synthesis of Inosine monophosphate, which is then differentiated by the action of the enzymes ADSS and ADSL, or IMPDH1 and GMPS, respectively. Much like the other metabolic pathways we have studied in this course, studies have recently shown connections between metabolism of purines and the progression of cancer. A 2017 article published by Wang et al., reports evidence that de novo purine biosynthesis, meaning the production of Purines from scratch, plays an important role in the pathogenesis of Glioblastoma, a type of primary brain tumor that is extremely malignant, and mostly incurable.
The authors of this article specifically focus on a type of cell called Brain Tumor Initiating Cells (BTICs) which are essentially cancer stem cells. BTICs are particularly problematic as they are extremely resilient to common treatments such as chemotherapy and radiation. Furthermore, they have a role in promoting the development of tumors and their infiltration into brain tissue. As we learned in Unit Two, cancer cells, including brain tumors, perform what is known as the Warburg effect. This refers to their tendency to derive most cellular energy from glycolysis, and use the intermediates from glycolysis for the construction of biomass rather than channeling them into the TCA cycle for further energy production. Studies have also shown that BTICs exhibit increased expression of the GLUT3 Transporter protein, part of a family of proteins that enable the passage of glucose across the cellular membrane. GLUT3 has a higher affinity for glucose than other related transporters, and thus the overexpression of this protein results in an increased influx of glucose into BTICs in comparison to other cells. Furthermore, patient data has indicated that high levels of GLUT3 and glucose in BTICs is correlated with a decreased lifespan.
Knowing that glucose serves as the primary carbon source for cancer cell biosynthesis, the authors isolated BTICs, and treated them with radiolabeled (C13) glucose. They then tracked the isotopic carbon in order to determine what pathways glycolytic intermediates were implicated in. The authors found that most glucose-derived carbons are incorporated into Guanine, Adenine, Inosine monophosphate (IMP), Adenosine monophosphate (AMP), and Guanosine monophosphate (GMP), all of which are products of purine biosynthesis. Furthermore, the authors report high levels of hypoxanthine, a molecule produced during the degradation of purines. This indicated that inhibition of the Purine degradation system could not be responsible for the observed increases in Guanine, Adenine, IMP, AMP, and GMP. Thus, they chose to further investigate the role of purine anabolism in the growth and proliferation of BTICs/glioblastoma. In order to do this, they focused on three sets of enzymes involved in purine biosynthesis. These targets were, phosphoribosyl pyrophosphate synthetase 1 (PRPS1) and phosphoribosyl pyrophosphate amidotransferase (PPAT) which are involved in the conversion of Ribose-5-Phosphate into IMP. They also targeted the previously mentioned ADSS and ADSL, responsible for converting IMP to AMP, as well as IMPDH1 and GMPS, responsible for converting IMP to GMP. For the purposes of controlling their experiments, the authors used Dentate Granule Cells (DGCs), from the dentate gyrus of the hippocampus as the normal cell type. Immunoblot analysis revealed elevated levels of all six de novo purine synthesis enzymes in BTICs relative to DGCs. These abnormally high protein levels indicated that this pathway may be vital to the development of glioblastomas from BTICs. Thus, they treated BTIC models with three different purine synthesis inhibitors: mycophenolic acid, mycophenolate mofetil and Ribavirin. All three of these drugs drastically decreased the proliferation of BTICs while having no impact on the growth of DGCs.
In order to further confirm the contribution of glycolysis/Warburg effect to Purine Biosynthesis, the authors developed experimental conditions that equated to short-term glucose restriction, long-term glucose restriction, and normal tumor growth conditions, and measured both mRNA expression and protein levels. The results showed decreases in expression and protein levels of all six purine biosynthetic enzymes under short-term glucose restriction. They then inhibited glycolysis by treatment with 2-deoxyglucose for two days. Inhibition of glycolysis resulted in a 50-80% decrease in mRNA levels, and decreases in the enzymes PRPS1, ADSL, and GMPS, as well as the metabolites IMP, AMP and GMP. In a subsequent experiment, they knocked out the gene for GLUT3 by more than 80%, which resulted in decreases in the levels of PRPS1, ADSL and GMPS. All of these findings indicate that glucose is used by BTICs as a source of carbon for de novo purine synthesis.
In another set of experiments, the authors set out to explore the effect of interfering with de novo purine synthesis on the survival of mouse models of glioblastoma. To do so, they first knocked out the genes encoding ADSL and GMPS, both of which had been found in elevated levels in 542 cases of glioblastoma. Upon knockdown of these genes, they found that the BTICs began to differentiate (a good thing!) leading to improved survival rates. They then knocked out the gene encoding PRPS1, the first enzyme of the de novo biosynthesis pathway, which has also been shown to be overexpressed in glioblastoma. The results again revealed increased survival rates, and BTIC differentiation.
Results found in Glioma databases showed intensely elevated levels of the purine synthesis enzymes in grade three and four gliomas, which have extremely poor prognoses. Therefore, the findings of this article are extremely important, as they reveal that inhibition of the de novo purine biosynthesis pathway could serve as a potential treatment for previously untreatable brain tumors. Furthermore, the inhibitors that they tested are all either currently, or in the process of being approved for clinical use in the treatment of other conditions. Thus, glioblastomas may be treatable using pre-existing drugs rather than requiring the development of new ones.
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