The elegant complexity of mammalian ecto-5'-nucleotidase (CD73)

Abstract

Purinergic signaling is a fundamental mechanism used by all cells to control their internal activities and interact with the environment. A key component of the purinergic system, the enzyme ecto-5'-nucleotidase (CD73) catalyzes the last step in the extracellular metabolism of ATP to form adenosine. Efforts to harness the therapeutic potential of endogenous adenosine in cancer have culminated in the ongoing clinical development of multiple CD73-targeting antibodies and small-molecule inhibitors. However, recent studies are painting an increasingly complex picture of CD73 mRNA and protein regulation and function in cellular homeostasis, physiological adaptation, and disease development. This review discusses the latest conceptual and methodological advances that are helping to unravel the complexity of this important enzyme that was identified nearly 90 years ago.

Keywords: adenosine; cancer; digestive system; inflammation; purinergic system; zonation.

Figure 1.. CD73 is an Essential Component of Purinergic Signaling and a Disease Target.

CD73 is a ubiquitously expressed ecto-nucleotidase of the purine metabolism pathway. As a glycosylphosphatidylinositol (GPI) -anchored glycoprotein on the plasma membrane, CD73 works in tandem with ectonucleoside triphosphate diphosphohydrolase-1 (CD39), which breaks down adenosine triphosphate (ATP) to form adenosine 5’-monophosphate (AMP) in a two-step process. Alternatively, AMP can be generated via direct conversion from ATP by the enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1). CD73 is the major enzyme that dephosphorylates AMP to generate extracellular adenosine (Ado), but this reaction can also be carried out by tissue non-specific alkaline phosphatase (TNAP) or prostatic acid phosphatase (PAP). CD73-generated adenosine directly exerts tissue-specific functions by binding to four different types of G-coupled adenosine receptors (AdoR), which regulate oxygen supply/demand ratios, inflammation and angiogenesis in a receptor-dependent manner. Additionally, adenosine is transported into the cytoplasm through equilibrative and concentrative nucleoside transporters (ENTs and CNTs). Due to its role in inflammatory responses and tumor growth and metastasis, small molecule inhibitors and monoclonal antibodies against CD73 are currently being tested in clinical trials for cancer immunotherapy and COVID-19 therapy.

Figure 2.. Zonal Expression of CD73 Supports Tissue-Specific Homeostasis.

A hypoxic environment induces CD73 expression in the apical membrane of different tissues. In the intestinal epithelia, for example, CD73 is present on villus tip enterocytes, which are subjected to low oxygen conditions. The villus tips face the intestinal lumen where anaerobic bacteria normally reside. Similarly, CD73 is zonally expressed in hepatocytes in the liver. In the hepatic lobule, periportal hepatocytes are located next to the portal triad on one side (encompassing the hepatic artery, vein, and bile duct), while pericentral hepatocytes reside adjacent to the central vein. This arrangement follows the oxygen gradient: highly oxygenated blood enters the liver via the hepatic artery from the portal triad, mixes with deoxygenated blood through the sinusoids between hepatocytes and is returned to the circulation via the central vein.

Figure 3.. Molecular Regulation of CD73.

The important functions of CD73 across cell- and tissue-types warrant different levels of molecular regulation. 1) At the transcriptional level, the expression of the CD73-encoding gene NT5E is upregulated by transcription factors (TF) such as HIF1α, SP1, SMAD, and AP1 and several micro RNAs (miRNAs), as noted in the text. In the context of cancer, NT5E mRNA undergoes 2) post-transcriptional splicing to generate an alternative NT5E-2 transcript or the circular RNA circNT5E. Subsequently, NT5E-1 mRNA is translated into CD73 and NT5E-2 mRNA into CD73S, which is an intracellular enzymatically-inactive isoform that targets canonical CD73 for proteosomal degradation. The circNT5E transcript is oncogenic and expressed in glioma and lung cancer. 3) At the post-translational level, CD73 protein is modified by the addition of a glycosylphosphatidylinositol (GPI)-anchor and by asparagine (N)-glycosylation in the endoplasmic reticulum (ER) and Golgi apparatus. The mature CD73 protein dimerizes and is transported to the plasma membrane facing the extracellular space. CD73 can be cleaved by phospholipase C (PLC) or matrix metallopeptidase 9 (MMP9) to generate a soluble form of the protein.

Figure 4.. New Tools to Study CD73 Regulation and Function.

The ubiquitous nature of CD73 and the purinergic signaling complexity conceal important tissue-specific mechanisms, which warrants development of new tools to study this ecto-enzyme. (Top Row) Recently synthesized fluorescent probes and small molecule inhibitors were designed based on the lead structure of the most common CD73 inhibitor, adenosine 5’-(α,β-methylene)diphosphate or APCP. These newer probes exhibit higher potency while also enabling visualization and monitoring of CD73. Additionally, studies demonstrating pro-tumorigenic functions of CD73 led to the advent of new monoclonal antibodies tested in clinical trials. In contrast, promoting CD73 activity may alleviate inflammation and platelet aggregation. To that end, a CD39-CD73 fusion protein was shown to sequentially hydrolyze pro-inflammatory ATP to anti-inflammatory adenosine. (Middle Row) To interrogate relevant disease mechanisms, patient-derived induced pluripotent stem cells (iPSCs) have become a robust model system. For example, fibroblasts derived from patients with a rare genetic mutation of CD73 can be reprogrammed to generate iPSCs. These, in turn, can be differentiated into affected cell types to study pathological mechanisms the rare disease Arterial Calcification Due to Deficiency of CD73 (ACDC). (Bottom Row) To elucidate tissue-specific functions of proteins, reporter mouse lines and targeted gene deletion have been instrumental. A new reporter mouse line called CD73-EGFP enables tracking cell lineage and identification of CD73⁺ cells. Another useful model is the floxed CD73 mouse line, which enables targeted deletion of CD73 in specific tissues when mated with Cre recombinase mice. Specifically, deletion of CD73 in the liver, intestines, and kidney demonstrated tissue-specific protection under physiological and pathological conditions.