As the basic components of RNA and DNA, purine nucleotides play an important role in cellular material metabolism and energy metabolism. For this reason, cells need an adequate supply of purine nucleotides to meet the needs of growth and proliferation. However, we still do not know how normal or malignant tumor tissues maintain nucleotide metabolic balance.
The Synthesis of purine nucleotides in vivo is currently believed to have two main pathways: de novo synthesis and salvage synthesis. The de novo synthesis pathway uses glutamine, aspartic acid and glycine to synthesize purine rings from scratch, and this process consumes a total of 6 ATP. This pathway is finely regulated by a variety of mechanisms, including transcriptional and post-transcriptional regulation, feedback inhibition regulation, multi-enzyme complex assembly processes, and some growth-promoting signaling pathways to support cell growth. The salvage synthesis pathway, as the name suggests, uses existing purine rings from diet or metabolism to synthesize Nucleotides, and this process only consumes 1 ATP. The salvage synthesis pathway is mainly regulated by two key enzymes, namely adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HPRT1), which convert adenine, hypoxanthine, and guanine into Adenosine monophosphate (AMP), inosine monophosphate (IMP), and guanosine monophosphate (GMP), respectively.
The traditional view is that differentiated cells mainly rely on salvage pathways to obtain nucleotides, while proliferating cells rely more on de novo synthesis pathways to meet the strong demand for nucleotides for proliferation. However, these views have not been proven by strong experimental evidence, and the role of these pathways in cancer is still unknown. Recently, the research team of Gerta Hoxhaj from the University of Texas Southwestern Medical Center published a paper in Cell titled "De novo and salvage purine synthesis pathways across tissues and tumors", revealing the unique role of purine salvage pathways in normal and tumor tissues that has not been valued.
The researchers used isotope-labeled nutrients to inject C57 mice and tracked changes in the de novo and salvage pathways by measuring and analyzing the concentrations of labeled metabolites in tissues and Tumors.
First, the researchers used [γ-15N]-glutamine or [γ, α-15N]-glutamine, a key raw material for de novo synthesis, to inject and label the activity of the de novo purine synthesis pathway. The labeling results showed that the small intestine showed the highest concentration of newly synthesized purine nucleotides, that is, it had the highest de novo purine synthesis activity.
Next, the researchers used [15N5]-adenine, [15N5]-adenosine, [13C5]-hypoxanthine, [15N4]-inosine, [15N5]-guanine, or [15N5]-guanosine, etc. to measure the activity of the salvage synthesis pathway. The results showed that for [15N5]-adenine, most tissues showed high enrichment of adenine and newly synthesized AMP and IMP, especially the kidney, lung, spleen, and small intestine. The heart and pancreas had low activity in salvage synthesis of adenine. For [15N5]-adenosine, the lung and spleen had high utilization activity.
[15N4]-inosine showed a similar enrichment pattern to adenine, while [13C5]-hypoxanthine, [15N5]-guanine, or [15N5]-guanosine had low enrichment in the circulation. However, regardless of the salvage metabolic raw material, the kidney showed a high salvage pathway activity. The in vitro kidney slice culture experiment further demonstrated the important role of the kidney in the salvage synthesis of purines in vivo.
For the low enrichment of [13C5]-hypoxanthine, [15N5]-guanine, or [15N5]-guanosine in the circulation, after excluding the cause of administration, the researchers speculated that it might be the rapid clearance of these precursors by the kidney, that is, degradation to uric acid through the action of xanthine dehydrogenase (XDH). Subsequent experimental results also demonstrated the important role of XDH-mediated degradation in regulating the salvage synthesis of purines in vivo.
The researchers further applied the above Labeling system to tumor model studies, including xenograft and transgenic mouse tumor models. The results showed that [γ-15N]-glutamine was utilized by all xenograft tumors and showed higher de novo synthesis activity than intestinal tissue. In terms of salvage synthesis, although key raw materials for salvage synthesis pathways such as [15N5]-adenine, [15N5]-adenosine, [13C5]-hypoxanthine, [15N4]-inosine, [15N5]-guanine, or [15N5]-guanosine all showed low enrichment in tumors, the salvage pathway activity (proportion of newly synthesized purine nucleotides) was even higher than the de novo synthesis activity. The researchers also verified the above conclusions in chemically induced colorectal cancer models and MyC overexpression-induced liver cancer models, and also verified the enhancing effect of MYC on the de novo synthesis pathway in tumors.
The above data have demonstrated that tumors effectively utilize the salvage synthesis pathway, but whether salvage synthesis is necessary for tumor growth remains unclear. The researchers further studied the key enzymes of the salvage synthesis pathway, including purine nucleic acid synthetases HPRT1 and APRT. Human breast normal and cancer tissue microchip samples demonstrated the relatively high expression of these enzymes in tumor tissues. Further researchers used CRISPR-Cas9 to Knock Out HPRT1 and APRT and knock out the de novo synthesis key enzyme GART in tumor cell lines and in vivo tumor models. The results showed that both HPRT1 and APRT significantly inhibited tumor growth. The impact of knocking out GART is even more significant.
The researchers finally demonstrated that supplementation of additional purine and pyrimidine nucleotides in food (i.e., enhanced intake of salvage synthetic raw materials) promoted tumor growth. Inhibition of nucleic acid transporters reversed this phenomenon.
Fig. 1 De novo and salvage purine synthesis pathways across tissues and tumors. (Tran, et al., 2024)
In short, this study deepened our understanding of purine nucleotide metabolism in normal and cancerous tissues, and emphasized the important role of salvage synthesis pathways in tumor growth. It provides new ideas for overcoming the resistance of de novo synthesis inhibitors and targeted eradication of tumors in clinical practice.
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