Nucleotide metabolism in the regulation of tumor microenvironment and immune cell function

Nucleotide metabolism plays a crucial role in the regulation of the tumor microenvironment (TME) and immune cell function. In the TME, limited availability of nucleotide precursors due to increased consumption by tumor cells and T cells affects both tumor development and immune function. Metabolic reprogramming in tumor cells favors pathways supporting growth and proliferation, including nucleotide synthesis. Additionally, extracellular nucleotides, such as ATP and adenosine, exhibit dual roles in modulating immune function and tumor cell survival. ATP stimulates antitumor immunity by activating purinergic receptors, while adenosine acts as a potent immunosuppressor. Targeting nucleotide metabolism in the TME holds immense promise for cancer therapy. Understanding the intricate relationship between nucleotide metabolism, the TME, and immune responses will pave the way for innovative therapeutic interventions.


Introduction
The tumor microenvironment (TME) is a heterogeneous ecosystem containing tumor cells, various stromal cells, blood vessels, and immune cells together with a range of other host cells [1].In the evolving tumor, the TME is an essential modulator of immune evasion and subsequent risk of tumor growth and metastasis.This modulation is based on the complex and multifaceted interaction between cells of the TME.In recent years, emerging evidence has demonstrated the nucleotide metabolism of both tumor cells and immune cells as targets and mediators of immune evasion, tumor growth, and metastasis in the TME.Here, we will review both the intracellular and extracellular nucleotide players and nucleotide pathways that are important for the TME and shortly discuss future perspectives in modulating these to efficiently combat cancer.

Role of intracellular nucleotides of cells in the tumor microenvironment
Both tumor cells and immune cells have a requirement of nucleotides that exceeds that of other cell types [2,3].Nucleotide synthesis requires the availability of various precursors, including amino acids, glucose, and nucleobases.In the TME, both cancer cells and immune cells rely on the same resources to meet their metabolic needs.As a result, substrates crucial for nucleotide synthesis are often contested in this environment [4].Notably, glutamine and glucose have frequently been described in the literature as limited in the TME due to increased consumption by dividing cancer cells and T cells [2].Glutamine is a vital nitrogen donor for de novo nucleotide biosynthesis and is necessary for the synthesis of purine rings and pyrimidine backbone.Glucose, on the other hand, is a major carbon source that fuels various metabolic pathways, including de novo nucleotide synthesis.Interestingly, recently it has been demonstrated that T cells and myeloid cells take up significantly more glucose in the TME than cancer cells.In contrast, cancer cells exhibited the highest uptake of glutamate [5].Other studies have demonstrated that cancers grown in glucose-deprived conditions can uptake and catabolize uridine for carbon metabolism as a substitute [6,7].These findings suggest a more complex interaction between tumor cells and immune cells in the TME that goes beyond simple competition and highlights a metabolic reprogramming in tumor cells that favors pathways that support enhanced growth and proliferation [2,4,8].Such regulatory pathways include aerobic glycolysis, glutaminolysis, and mitochondrial biogenesis and activities [9][10][11].This reprogramming further extends to specific pathways involved in nucleotide metabolism.Here, increased activities of enzymes related to salvage and de novo nucleotide synthesis as well as decreased activity of enzymes related to the breakdown of nucleotides are linked to tumor growth and metastasis [2,4,11].
The salvage pathway allows cells to recycle nucleotides and nucleobases from the intra-and, via uptake, the extracellular environment.Even though immune cells and cancer cells are predisposed to prefer de novo nucleotide synthesis [12], components of the salvage pathway have nevertheless been demonstrated to be related to cancer development.The enzyme hypoxanthine guanine phosphoribosyltransferase (HPRT) is a salvage pathway enzyme responsible for the formation of inosine monophosphate (IMP) and guanosine monophosphate (GMP) from precursors within the cell to eventually form inosine and guanine.HPRT is an established biomarker for evaluation of mutagenesis in vitro and in vivo [13] and has been found overexpressed in histological samples from lung, colon, prostate, and breast cancer [14].Thymidine kinase 1 (TK1) mediates the conversion of thymidine to deoxythymidine monophosphate (dTMP) in the salvage pathway and thymidylate synthase (TYMS) catalyzes the conversion of deoxyuridine monophosphate to dTMP.TK1 and TYMS have been established as cancer biomarkers for multiple cancers, including leukemia, colorectal cancer, lung cancer, breast cancer, and prostate cancers [15,16].Whereas the salvage pathway is an energy-efficient process, rapidly dividing cancer cells often rely more heavily on the de novo pathway to meet demand for nucleotides [17].Here, phosphoribosyl pyrophosphate synthetase, GMP synthase, phosphoribosyl pyrophosphate amidotransferase, carbamoyl-phosphate synthetase-2 aspartate transcarbamylase dihydroorotase, ribonucleotide reductase (dihydroorotate dehydrogenase) (DHODH), inosine monophosphate dehydrogenase (I and II), hexokinase, transketolase, ribose-5phosphate isomerase-A (RPIA), methylene-THF dehydrogenase/cyclohydrolase, and others have been clearly described in literature to be overactivated in cancers [16,18,19].
Nucleotides, the building blocks of DNA and RNA, serve not only as genetic building blocks, but also as key regulators of intracellular signaling pathways and as substrates for other essential biosynthetic pathways.One such pathway is the cyclic GMP-AMP synthase (cGAS) stimulator of interferon gene (STING) signaling, initiated by excessive cytoplasmic double-stranded DNA [20,21].Since genome instability is a hallmark of cancer development, the release of various forms of nuclear DNA into the cytoplasm is often reported [22], and can lead to an innate immune response via cGAS-STING.Therefore, cGAS-STING activation is suggested as an anticancer therapy [23], and downstream signaling is often impaired in different types of cancers, including gastric and colon cancers [24].However, tumor development and the TME are highly complex and their link to cytoplasmic DNA signaling seems to be dependent on tumor stage and type [25].Upregulated nucleotide metabolism in cancer cells has been demonstrated to induce increased expression of MHC class I polypeptide-related sequence A (MICA), facilitating better binding to natural killer group 2D receptor (NKG2D) expressed in NK cells and inducing proliferation of NK cells and immune response [26].In contrast, inhibition of partial pyrimidine synthesis in cancer cells results in increased tumorigenesis and lysosome accumulation [27].In T cells, an inhibition of the DHODH has been demonstrated to inhibit replication, but not activation of T cells.This response was not related to synthesis of RNA or DNA.Replication potential was fully restored in cells supplemented with uridine, and led the authors to suggest that the nucleotides play a novel regulatory role in the T cells [3].

Role of extracellular nucleotides in the tumor microenvironment
In the milieu of the TME, extracellular nucleotides and their derivatives function in dual roles as both potent activators and inhibitors of immune function [28,29] as well as tumor cell survival and metastasis.Adenine nucleotides are found in the TME in high millimolar concentrations that are several exponential folds higher than the low and constant nanomolar levels of the nucleotides found in the extracellular space under nonpathological conditions [30,31].Nucleotides can be released into the TME as a result of necrosis, released from stressed cells during ischemia, hypoxia, and inflammation, and be released through membrane transporters and vesicular exocytosis [32][33][34].Extracellular nucleotides and nucleosides are known to be recognized by three different families of purigenic receptors expressed in different combinations on most mammalian cells, namely P1, P2X, and P2Y [29,35,36].The activation of the different purigenic receptors can stimulate multiple signaling pathways, leading to diverse effects depending on the cell type and receptor subtype.Upon engagement of the purigenic receptor subtypes P2X and P2Y on immune and endothelial cells, extracellular ATP acts as a danger signal involved in promoting the recruitment of innate immune cells and in the priming of antitumor immunity [37,38].This response includes the activation of T cells and the promotion of Th17 cell differentiation, triggered by the induction of IL-23 and IL-1β from myeloid cells and monocyte-macrophages. ATP inhibits the function of Tregs, Tr1 cells, and B cells, while favoring the chemotaxis of NK cells and neutrophils.Moreover, ATP promotes the differentiation of macrophages toward an inflammatory phenotype and induces the maturation of dendritic cells [30].In tumor cells, activation of P2X and P2Y receptors by extracellular ATP results in cytotoxicity [39][40][41], but has also been correlated with tumor growth and, in the setting of prostate, renal and breast cancers, especially P2Y activation has been associated with invasiveness, metastasis, and spreading [42][43][44][45][46].
In contrast to extracellular ATP, extracellular adenosine is generally acknowledged to be a potent immunosuppressor [47].Adenosine is recognized by the purinergic receptor P1 that is widely expressed by immune cells.Activation of these receptors results in an elevation of intracellular cyclic AMP levels, which reduces the production of pro-inflammatory mediators, and increases the production of anti-inflammatory factors in immune cells [48].The P1 receptor has also been described to be overexpressed in human hepatocellular, colorectal, oral squamous, and bladder urothelial carcinomas [28], where the presence of adenosine has been shown to stimulate migration and proliferation of some types of tumors but inhibit others.
In the extracellular environment, ATP can undergo hydrolysis by plasma membrane nucleotidases, leading to the formation of adenosine diphosphate (ADP), adenosine monophosphate (AMP), and eventually adenosine, thereby changing its role from an immunostimulant to an immunosuppressant.Among these ectonucleotidases, two have emerged in the literature as particularly crucial in the context of the TME: CD39 and CD73 [30].These enzymes are expressed on various immune cell types, including B cells, monocytes, and CD4+ and CD8+ T cells, with a somewhat lesser degree of expression on NK cells [30].However, it is worth noting that CD39 and CD73 are also markedly expressed in several human tumor types.These ectonucleotidases have been identified in renal cell carcinoma, ovarian cancer, sarcoma cancer, breast cancer, lymphoma, bladder cancer, colon cancer, and melanoma [49,50].The presence of CD39 and CD73 in the TME is associated with several poor prognostic factors and clinical outcomes.High expression levels of CD39 and CD73 have been correlated with advanced tumor stage, increased metastasis, and reduced overall survival in cancer patients.Furthermore, studies have shown that the combined expression of CD39 and CD73 is associated with an even worse prognosis compared with their individual expression levels [49,50].Additionally, recent studies have unveiled the presence of CD39 and CD73 in tumor-derived exosomes, accompanied by elevated levels of adenosine and other purines [51].This discovery suggests the existence of a shuttle mechanism facilitating local and distant signaling, ultimately bolstering tumor growth while concurrently suppressing immune cell functions [51].
Other purines have also shown to have immunosuppressive attributes.Dendritic cells have been demonstrated to release inosine that dampens T-cell activation and thereby protects against excessive antiviral T-cell responses, autoimmunity, and autoinflammation [52].

Conclusions and perspectives
With the importance of both intracellular and extracellular nucleotides for both immune cells and tumor cells, targeting nucleotide metabolism in the TME holds significant potential for cancer therapy.However, this is not a new approach, and many drugs inhibiting parts or whole of the nucleotide metabolism have been proposed and used as potent anticancer drugs for the last 70 years.Methotrexate was developed in the 1940s and has been used for the treatment of various cancers, including leukemia, lymphoma, and solid tumors [53].Methotrexate inhibits dihydrofolate reductase, an enzyme involved in the conversion of dihydrofolate to tetrahydrofolate, thereby disrupting the availability of folate, a crucial cofactor in nucleotide synthesis.The problem with methotrexate, and most drugs with similar function, is that they also target the metabolic processes of normal cells, causing side effects [54].Furthermore, many of the targets affected by this group of drugs, also target functionality of immune cells, and many of the drugs that were first introduced as chemotherapeutic drugs have found uses as immune suppressants in autoimmunity diseases [55].Nevertheless, today, there are over 20 approved nucleotide and nucleotide analogs used in cancer chemotherapies, which account for nearly 20% of all drugs in cancer treatment [11].As discussed in this review, the diversity and complex interaction between tumor cells and immune cells impacting each other's intracellular and extracellular nucleotide metabolism and sensitivity necessitates a more personalized approach.Inhibition of one part of the nucleotide metabolism may inhibit some tumors, while promoting others.Targeting the nucleotide metabolism in TME is however still extremely promising and has been suggested by several to be extremely important in future immunotherapies [11].By modulating nucleotide availability and metabolic pathways, it is possible to overcome immunosuppression, enhance immune cell function, and potentially overcome therapeutic resistance.Continued research and clinical investigations are needed to fully explore the potential of targeting nucleotide metabolism in combination with immunotherapies and to identify optimal strategies for improving patient outcomes in cancer treatment.