Unveiling the Role of CD73 A Key Player in Immune Regulation and Beyond

What is CD73?

CD73, also known as ecto-5′-nucleotidase, is a groundbreaking immunoinhibitory protein that assumes a pivotal role in tumor growth and metastasis. Its primary function revolves around converting extracellular ATP into immunosuppressive adenosine, working in tandem with CD39 within normal tissues to curtail excessive immune reactions. However, this mechanism is often hijacked by tumors, utilizing CD73 to foster an adenosinergic process that shields them from immune assaults.

Notably, both cancer cells and host cells employ inducible CD73 expression, alongside other adenosinergic molecules, to establish and perpetuate an immunosuppressive microenvironment within the tumor. This orchestrated strategy affects various dimensions of the immune response. Given its multifaceted contribution to tumor advancement as an emerging immune checkpoint, CD73 stands out as a promising target for cancer treatment. This is especially true when combined with traditional therapies and/or other immune checkpoint inhibitors, opening up new avenues for comprehensive cancer intervention.

Exploring the Structure and Function of CD73

The CD73 gene finds its home in the 6q14.3 region of human chromosome 6, encoding a protein comprised of 549 amino acid residues. CD73 functions as a glycosylphospholipase, residing on the cell membrane surface and engaging with the extracellular matrix through its extracellular C-terminal tail domain. The protein's structure can be broken down into three key elements: a signal peptide sequence, an extracellular catalytic domain, and an intracellular phospholipid peptide binding domain.

CD73, also known as membrane-bound ecto-5′-nucleotidase NT5E, performs the crucial task of breaking down extracellular adenosine monophosphate (AMP) into adenosine and inorganic phosphate (P). Before reaching NT5E, adenosine triphosphate (ATP) undergoes a two-step hydrolysis process, transforming into AMP thanks to the enzyme ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1), also known as CD39.

The adenosine produced through these processes exerts potent anti-inflammatory effects. It achieves this by binding to the adenosine A2A receptor (ADORA2A), which is expressed by various immune cells like T cells, natural killer (NK) cells, and dendritic cells (DCs). This binding triggers a cascade involving cAMP, effectively blocking the effector functions of these immune cells. Notably, adenosine's influence also extends to the A2B receptor (ADORA2B), found on DCs and macrophages. These cells are similarly suppressed by adenosine.

Intriguingly, cancer cells exploit this mechanism to evade immune surveillance by ramping up NT5E protein levels. Additionally, adenosine binds to the A2B receptor expressed by cancer cells themselves, facilitating tumor cell survival and proliferation. Further complexity arises as cancer cells also express the adenosine A1 receptor (ADORA1) and A3 receptor (ADORA3). The binding of adenosine to these receptors triggers tumor cell migration and proliferation through signaling pathways involving Gαi proteins.

Beyond its immune-modulating functions, adenosine plays a role in adapting to hypoxic conditions and exhibits pro-angiogenic potential, further influencing the tumor microenvironment.

CD73 and Adenosine Receptor Activity Promotes Immunosuppression

Ecto-5′-nucleotidase (NT5E), commonly referred to as CD73, emerges as a central player in generating extracellular adenosine. When tissues face damage, inflammation, or hypoxic stress, adenosine triphosphate (ATP) is released from stressed, necrotic, or apoptotic cells. This ATP undergoes a stepwise transformation, first by ectonucleoside triphosphate diphosphohydrolase-1 (CD39), converting ATP into adenosine monophosphate (AMP), and then by CD73, converting AMP into extracellular adenosine.

ATP initially triggers inflammation through ATP receptors. However, the subsequent conversion of ATP into extracellular adenosine and the activation of adenosine receptors serve to temper this inflammation[1-3]. Extracellular adenosine communicates through four adenosine receptors: A1R, A2AR, A2BR, and A3R[4]. The earliest links between extracellular adenosine and immunosuppression were established through studies on methotrexate's anti-inflammatory activity[5]. Seminal research further revealed that A2AR signaling plays an essential role in quelling tissue-damaging inflammation[6].

Extracellular adenosine provides protective effects by mitigating inflammation in conditions such as myocardial injury[7], acute lung injury[8], intestinal ischemia-reperfusion injury[9], and inflammatory bowel disease[10]. In the context of cancer, tumors harness extracellular adenosine to shield cancer cells. Adenosine accumulates within tumors and hinders the activity of cytotoxic T cells and natural killer cells. Numerous studies employing syngeneic and spontaneous tumor models have demonstrated that genetic deletion or pharmacological inhibition of CD73 or A2AR significantly reduces tumor growth and metastasis. This effect is primarily attributed to the restoration of antitumor immunity. Moreover, these interventions enhance the sensitivity of these mice to chemotherapy[11] and curtail angiogenesis. Correspondingly, elevated CD73 expression is frequently observed in human tumors and is associated with a poor prognosis. CD73 is also implicated in drug resistance, epithelial-to-mesenchymal transition (EMT), and the proliferation and stemness of cancer cells[12].

Tumors also exhibit slower growth in mice lacking A2BR or when treated with A2BR antagonists. For the most part, the activation of A2AR, and to a lesser extent A2BR, on various types of immune cells fosters immunosuppression.

CD73 Expression and Its Impact on Tumor Cells

CD73 is found at higher expression levels in the majority of human solid tumors, and its presence is closely linked to tumor invasiveness and metastasis[15]. Research[16] has confirmed that the extracellular adenosine produced by CD73 on tumor cells plays a significant role in immune evasion, facilitating tumor growth and metastasis. To better understand the role of CD73 in tumorigenesis, multiple CD73-deficient tumor models have been studied, shedding light on its importance in both tumor cells and host cells[17].

Beyond its immune-regulating functions, CD73 influences various aspects of tumorigenesis, including proliferation, adhesion, migration, angiogenesis, and metastasis. It promotes tumor cell proliferation by regulating the cell cycle, apoptosis, and signaling pathways like EGFR, β-catenin/cyclin D1, VEGF, and AKT/ERK[18]. Interestingly, CD73, aside from its enzymatic function, also facilitates cell-to-cell adhesion, migration, invasion of cancer cells, and promotes stemness[19]. Notably, CD73, whether on tumor cells or host cells, plays a crucial role in tumor angiogenesis. Furthermore, studies highlight the significance of CD73-A2AR signaling in tumor-associated lymphangiogenesis[20], suggesting the potential use of adenosine-blocking agents to inhibit pathological lymphangiogenesis in cancers and prevent tumor dissemination.

Recent research has unveiled that cancer cell-intrinsic CD73 expedites metastasis through the PI3K/AKT signaling pathway and RICS/Rho GTPase signaling pathway. These findings correlate CD73 expression with worse prognosis[21] and poorer responses to therapeutic agents. However, it's important to note that CD73 isn't universally upregulated in all cancers; its expression can also be correlated with a positive prognosis. Aberrantly glycosylated CD73 and a human-specific CD73 isoform (CD73s)[22] have been identified in human hepatocellular carcinoma (HCC), leading to the functional suppression of tumor CD73. Additionally, CD73 expression was found to be downregulated in advanced-stage prostate, laryngeal, and high-grade colon carcinomas. Lower CD73 expression levels were observed in poorly differentiated and advanced-stage endometrial carcinomas compared to normal and well-differentiated, early-stage tumors, with higher CD73 expression associated with improved overall survival[23]. In early-stage endometrial tumors, CD73-generated adenosine has been shown to protect epithelial integrity via actin polymerization. Consequently, the role of CD73 in cancers appears to be complex, possibly due to its involvement in non-tumor-promoting effects mediated by CD73.

Navigating the Adenosinergic Signaling Pathway

The adenosinergic signaling pathway orchestrates intricate interactions within and outside cells. The extracellular milieu is governed by a web of molecules. ATP and ADP undergo hydrolysis, facilitated by CD39, which transforms them into AMP. The subsequent conversion of AMP to adenosine is managed by CD73. Extracellular adenosine assumes multiple roles: it triggers cellular signaling via adenosine receptors (ARs) through G proteins and associated effectors; it can degrade into inosine via ADA; or it can traverse the plasma membrane through either Na+-independent passive nucleoside transporters (ENTs) or Na+-dependent active nucleoside transporters (CNTs).

Within the cellular confines, the story continues. ATP undergoes hydrolysis, with NDPK and AK transforming it into ADP and AMP respectively. AMP within cells is then further refined into adenosine by cN. This internal adenosine encounters various fates: degradation into inosine through ADA, transportation to the extracellular realm, or resynthesis into ATP through metabolic enzymes.

Notably, the elevation of the AMP-to-ATP ratio prompts the activation of AMP-activated protein kinase (AMPK). This AMPK activation subsequently influences mitochondrial homeostasis, sculpting mitochondrial fission/fusion or mitophagy by modulating enzymes such as MFF or ULK1.

Even DNA methylation finds its place in this intricate dance. Intracellular adenosine comes into play, regulating DNA methylation by negatively impacting transmethylation pathways. Here, DNA methyltransferases (DNMTs) demethylate S-adenosyl-methionine (SAM) to S-adenosyl-homocysteine (SAH), which in turn boosts DNA methylation. The next step involves SAH's breakdown into adenosine and homocysteine, orchestrated by S-adenosyl-homocysteine hydrolase (SAHH). Interestingly, adenosine impairs SAHH activity, altering SAH levels and curbing SAM-dependent DNMT activity.

In this intricate narrative, a host of key players emerge: ADA (adenosine deaminase), ADK (adenosine kinase), AK (adenylate kinase), AMPK (AMP-activated protein kinase), ARs (adenosine receptors), CNTs (concentrative nucleoside transporters), ENTs (equilibrative nucleoside transporters), NDPK (nucleotide diphosphokinase), NT5C/cN (endonucleotidase), SAH (S-adenosyl-homocysteine), DNMTs (DNA methyltransferases), SAHH (S-adenosyl-homocysteine hydrolase), and SAM (S-adenosyl-methionine).

CD73 Protein

Recombinant Human CD73 Protein

Click here for more CD73

Synonym : 5' NT 5' nucleotidase (CD73) 5' nucleotidase precursor 5' nucleotidase, ecto 5' nucleotidase, ecto (CD73) 5'-NT 5'-nucleotidase 5NTD_HUMAN CD73 CD73 antigen E5NT Ecto 5' nucleotidase Ecto-5'-nucleotidase eN eNT NT NT5 NT5E NTE Purine 5 Prime Nucleotidase

References:

[1] Linden J, Koch-Nolte F, Dahl G. Purine release, metabolism„ and signaling in the inflammatory response. Annu Rev Immunol. (2019) 37:325–47. doi: 10.1146/annurev-immunol-051116-052406
[2] Bowser JL, Lee JW, Yuan X, Eltzschig HK. The hypoxia-adenosine link during inflammation. J Appl Physiol. (2017) 123:1303–20. doi: 10.1152/japplphysiol.00101.2017
[3] Bowser JL, Phan LH, Eltzschig HK. The hypoxia-adenosine link during intestinal inflammation. J Immunol. (2018) 200:897–907. doi: 10.4049/jimmunol.1701414
[4] Robeva AS, Woodard RL, Jin X, Gao Z, Bhattacharya S, Taylor HE, et al. Molecular characterization of recombinant human adenosine receptors. Drug Dev Res. (1996) 39:243–52. doi: 10.1002/(SICI)1098-2299(199611/12)39:3/4243::AID-DDR33.0.CO;2-R
[5] Cronstein BN, Naime D, Ostad E. The antiinflammatory mechanism of methotrexate. Increased adenosine release at inflamed sites diminishes leukocyte accumulation in an in vivo model of inflammation. J Clin Invest. (1993) 92:2675–82. doi: 10.1172/JCI116884
[6] Ohta A, Sitkovsky M. Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage. Nature. (2001) 414:916–20. doi: 10.1038/414916a
[7] Rose JB, Naydenova Z, Bang A, Eguchi M, Sweeney G, Choi DS, et al. Equilibrative nucleoside transporter 1 plays an essential role in cardioprotection. Am J Physiol Heart Circ Physiol. (2010) 298:H771–7. doi: 10.1152/ajpheart.00711.2009
[8] Rose JB, Naydenova Z, Bang A, Eguchi M, Sweeney G, Choi DS, et al. Equilibrative nucleoside transporter 1 plays an essential role in cardioprotection. Am J Physiol Heart Circ Physiol. (2010) 298:H771–7. doi: 10.1152/ajpheart.00711.2009
[9] Schingnitz U, Hartmann K, Macmanus CF, Eckle T, Zug S, Colgan SP, et al. Signaling through the A2B adenosine receptor dampens endotoxin-induced acute lung injury. J Immunol. (2010) 184:5271–9. doi: 10.4049/jimmunol.0903035
[10] Willingham SB, Ho PY, Hotson A, Hill C, Piccione EC, Hsieh J, et al. A2AR Antagonism with CPI-444 induces antitumor responses, and augments efficacy to Anti-PD-(L)1, and Anti-CTLA-4 in preclinical models. Cancer Immunol Res. (2018) 6:1136–49. doi: 10.1158/2326-6066.CIR-18-0056
[11] Yan A, Joachims ML, Thompson LF, Miller AD, Canoll PD, Bynoe MS. CD73 promotes glioblastoma pathogenesis, and enhances its chemoresistance via A2B adenosine receptor signaling. J Neurosci. (2019) 39:4387–402. doi: 10.1523/JNEUROSCI.1118-18.2019
[12] Xiong L, Wen Y, Miao X, Yang Z. NT5E, and FcGBP as key regulators of TGF-1-induced epithelial-mesenchymal transition (EMT) are associated with tumor progression and survival of patients with gallbladder cancer. Cell Tissue Res. (2014) 355:365–74. doi: 10.1007/s00441-013-1752-1
[13] Kordaß T, Osen W, Eichmüller SB. Controlling the Immune Suppressor: Transcription Factors and MicroRNAs Regulating CD73/NT5E. Front Immunol. 2018 Apr 18;9:813. doi: 10.3389/fimmu.2018.00813. PMID: 29720980; PMCID: PMC5915482.
[14] Harvey JB, Phan LH, Villarreal OE, Bowser JL. CD73's Potential as an Immunotherapy Target in Gastrointestinal Cancers. Front Immunol. 2020 Apr 15;11:508. doi: 10.3389/fimmu.2020.00508. PMID: 32351498; PMCID: PMC7174602.
[15] Chen S, Wainwright DA, Wu JD, Wan Y, Matei DE, Zhang Y, Zhang B. CD73: an emerging checkpoint for cancer immunotherapy. Immunotherapy. 2019 Aug;11(11):983-997. doi: 10.2217/imt-2018-0200. Epub 2019 Jun 21. PMID: 31223045; PMCID: PMC6609898.
[16] Jin D, Fan J, Wang L, Thompson LF, Liu A, Daniel BJ, Shin T, Curiel TJ, Zhang B. CD73 on tumor cells impairs antitumor T-cell responses: a novel mechanism of tumor-induced immune suppression. Cancer Res. 2010 Mar 15;70(6):2245-55. doi: 10.1158/0008-5472.CAN-09-3109. Epub 2010 Feb 23. PMID: 20179192; PMCID: PMC2883609.
[17] Stagg J, Beavis PA, Divisekera U, Liu MC, Möller A, Darcy PK, Smyth MJ. CD73-deficient mice are resistant to carcinogenesis. Cancer Res. 2012 May 1;72(9):2190-6. doi: 10.1158/0008-5472.CAN-12-0420. Epub 2012 Mar 6. PMID: 22396496.
[18] Zhou L, Jia S, Chen Y, Wang W, Wu Z, Yu W, Zhang M, Ding G, Cao L. The distinct role of CD73 in the progression of pancreatic cancer. J Mol Med (Berl). 2019 Jun;97(6):803-815. doi: 10.1007/s00109-018-01742-0. Epub 2019 Mar 29. PMID: 30927045; PMCID: PMC6525710.
[19] Lupia M, Angiolini F, Bertalot G, Freddi S, Sachsenmeier KF, Chisci E, Kutryb-Zajac B, Confalonieri S, Smolenski RT, Giovannoni R, Colombo N, Bianchi F, Cavallaro U. CD73 Regulates Stemness and Epithelial-Mesenchymal Transition in Ovarian Cancer-Initiating Cells. Stem Cell Reports. 2018 Apr 10;10(4):1412-1425. doi: 10.1016/j.stemcr.2018.02.009. Epub 2018 Mar 15. PMID: 29551673; PMCID: PMC5998305.
[20] Allard B, Cousineau I, Allard D, Buisseret L, Pommey S, Chrobak P, Stagg J. Adenosine A2a receptor promotes lymphangiogenesis and lymph node metastasis. Oncoimmunology. 2019 Apr 26;8(8):1601481. doi: 10.1080/2162402X.2019.1601481. PMID: 31413909; PMCID: PMC6682371.
[21] Buisseret L, Pommey S, Allard B, Garaud S, Bergeron M, Cousineau I, Ameye L, Bareche Y, Paesmans M, Crown JPA, Di Leo A, Loi S, Piccart-Gebhart M, Willard-Gallo K, Sotiriou C, Stagg J. Clinical significance of CD73 in triple-negative breast cancer: multiplex analysis of a phase III clinical trial. Ann Oncol. 2018 Apr 1;29(4):1056-1062. doi: 10.1093/annonc/mdx730. PMID: 29145561; PMCID: PMC5913595.
[22] Snider NT, Altshuler PJ, Wan S, Welling TH, Cavalcoli J, Omary MB. Alternative splicing of human NT5E in cirrhosis and hepatocellular carcinoma produces a negative regulator of ecto-5'-nucleotidase (CD73). Mol Biol Cell. 2014 Dec 15;25(25):4024-33. doi: 10.1091/mbc.E14-06-1167. Epub 2014 Oct 8. PMID: 25298403; PMCID: PMC4263446.
[23] Bowser JL, Blackburn MR, Shipley GL, Molina JG, Dunner K Jr, Broaddus RR. Loss of CD73-mediated actin polymerization promotes endometrial tumor progression. J Clin Invest. 2016 Jan;126(1):220-38. doi: 10.1172/JCI79380. Epub 2015 Dec 7. PMID: 26642367; PMCID: PMC4701552.
[24] Chang, CP., Wu, KC., Lin, CY. et al. Emerging roles of dysregulated adenosine homeostasis in brain disorders with a specific focus on neurodegenerative diseases. J Biomed Sci 28, 70 (2021). https://doi.org/10.1186/s12929-021-00766-y.