CD73 Regulation Research Articles

5ʹ-Ectonucleotidase CD73/NT5E supports EGFR-mediated invasion of HPV-negative head and neck carcinoma cells

BackgroundEpithelial-to-mesenchymal transition (EMT) of malignant cells is a driving force of disease progression in human papillomavirus-negative (HPV-negative) head and neck squamous cell carcinomas (HNSCC). Sustained hyper-activation of epidermal growth factor receptor (EGFR) induces an invasion-promoting subtype of EMT (EGFR-EMT) characterized by a gene signature (“‘EGFR-EMT_Signature’”) comprising 5´-ectonucleotidase CD73. Generally, CD73 promotes immune evasion via adenosine (ADO) formation and associates with EMT and metastases. However, CD73 regulation through EGFR signaling remains under-explored and targeting options are amiss.MethodsCD73 functions in EGFR-mediated tumor cell dissemination were addressed in 2D and 3D cellular models of migration and invasion. The novel antagonizing antibody 22E6 and therapeutic antibody Cetuximab served as inhibitors of CD73 and EGFR, respectively, in combinatorial treatment. Specificity for CD73 and its role as effector or regulator of EGFR-EMT were assessed upon CD73 knock-down and over-expression. CD73 correlation to tumor budding was studied in an in-house primary HNSCC cohort. Expression correlations, and prognostic and predictive values were analyzed using machine learning-based algorithms and Kaplan–Meier survival curves in single cell and bulk RNA sequencing datasets.ResultsCD73/NT5E is induced by the EGF/EGFR-EMT-axis and blocked by Cetuximab and MEK inhibitor. Inhibition of CD73 with the novel antagonizing antibody 22E6 specifically repressed EGFR-dependent migration and invasion of HNSCC cells in 2D. Cetuximab and 22E6 alone reduced local invasion in a 3D-model. Interestingly, combining inefficient low-dose concentrations of Cetuximab and 22E6 revealed highly potent in invasion inhibition, substantially reducing the functional IC50 of Cetuximab regarding local invasion. A role for CD73 as an effector of EGFR-EMT in local invasion was further supported by knock-down and over-expression experiments in vitro and by high expression in malignant cells budding from primary tumors. CD73 expression correlated with EGFR pathway activity, EMT, and partial EMT (p-EMT) in malignant single HNSCC cells and in large patient cohorts. Contrary to published data, CD73 was not a prognostic marker of overall survival (OS) in the TCGA-HNSCC cohort when patients were stratified for HPV-status. However, CD73 prognosticated OS of oral cavity carcinomas. Furthermore, CD73 expression levels correlated with response to Cetuximab in HPV-negative advanced, metastasized HNSCC patients.ConclusionsIn sum, CD73 is an effector of EGF/EGFR-mediated local invasion and a potential therapeutic target and candidate predictive marker for advanced HPV-negative HNSCC.

CD73 mitigates hepatic damage in alcoholic steatohepatitis by regulating PI3K/AKT-mediated hepatocyte pyroptosis

BackgroundAlcohol use is a major risk factor for death and disability, resulting in a significant global disease burden. Alcoholic steatohepatitis (ASH) reflects an acute exacerbation of alcoholic liver disease (ALD) and is a growing health care and economic burden worldwide. Pyroptosis plays a central role in the pathogenesis of ASH. Nt5e (CD73) is a cell surface ecto-5ʹ-nucleotidase, which is a key enzyme that converts the proinflammatory signal ATP to the anti-inflammatory mediator adenosine (ADO). Studies have found that CD73 is involved in multiple diseases and can alleviate gasdermin D (GSDMD)-mediated pyroptosis; however, its role and mechanism in ASH are not explicit. AimTo investigate the role and mechanisms of CD73-mediated hepatocyte pyroptosis in alcohol-induced liver injury through in vivo and in vitro experiments. MethodsCD73 knockout (CD73-/-) mice, wild-type (WT) mice, and AML-12 cells were used to evaluate the effect of CD73 on hepatocyte pyroptosis in vivo and in vitro. A combination of molecular and histological methods was performed to assess pyroptosis and investigate the mechanism both in vivo and in vitro. ResultsThe protein expression of CD73 and pyroptosis pathway-associated genes was increased significantly in hepatocyte injury model both in vivo and in vitro. In vivo, CD73 knockout dramatically aggravated inflammatory damage, lipid accumulation, and hepatocyte pyroptosis in the liver. In vitro, overexpression of CD73 by pEGFP-C1/CD73 can decrease NLRP3 inflammasome activation and pyroptosis in hepatocytes. Further analysis revealed that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is a possible mechanism of CD73 regulation. Meanwhile, this pathological process was inhibited after the use of PI3K inhibitors. ConclusionOur results show a novel function of CD73 regulates hepatocytes pyroptosis and highlights the therapeutic opportunity for reducing the disease process in ALD.

Abstract P2-20-15: Proteolytic regulation of CD73 by TRIM2 orchestrates tumor immunogenicity

Abstract Proteolytic regulation of CD73 by TRIM2 orchestrates tumor immunogenicity Ziyi Fu1,2, Siqi Chen3, Yueming Zhu4, Donghong Zhang4, Ping Xie3, Qiao Jiao4, Shipeng Xu1, Yifan Xue6, Xinhua Lu6, Xinxin Song7, Massimo Cristofanilli8, William J Gradishar3, Kevin Kalinsky5, Yongmei Yin2, Bin Zhang3 and Yong Wan4,5* 1Department of Obstetrics and Gynecology, Department of Pharmacology, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, USA. 2Department of Oncology, The First Affiliated Hospital of Nanjing Medical University, China 3Department of Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, USA. 4Department of Pharmacology and Chemical Biology, Winship Cancer Institute, Emory University School of Medicine, USA. 5Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University School of Medicine, USA. 6Department of Biomedical Informatics, University of Pittsburgh School of Medicine, USA. 7Department of Surgery, UT Southwestern Medical Center, Dallas, TX, USA. 8Department of Medicine, Weill Cornell Medicine, USA. ABSTRACT Despite the rapid utilization of immunotherapy, emerging challenges to the current immune checkpoint blockade need to be resolved. Here, we report that uncontrolled elevation of CD73 levels due to its aberrant turnover is tightly correlated with poor prognosis in immune-cold triple negative breast cancers (TNBCs), which impedes the efficacy for chemotherapy and immunotherapy. We have identified TRIM21 as a E3 ligase that governs CD73 destruction. Disruption of TRIM21 stabilizes CD73 that in turn enhances CD73-catalyzed production of adenosine, resulting in the suppression of CD8+ T cell function. The immunostaining demonstrated the cytosolic colocalization between TRIM21 and CD73. Molecular mapping further identified the amino acid stretches from 340-476 on TRIM21 and residues from 176–224 on CD73 mediated the interaction between TRIM21 and CD73. Replacement of lysine 133, 208, 262 and 321 by arginine on CD73 attenuated CD73 ubiquitylation and degradation. Moreover, TRIM21 is upregulated but CD73 is downregulated in response to IFN- secreted from activated CD8+ T cells in a feedback manner. Importantly, in preclinical animal models, diminishing of CD73 ubiquitylation remarkably promotes tumor growth and impedes antitumor immunity. In addition, a TRIM2hight/CD73low signature in a subgroup of human breast malignancies was associated with a favorable immune profile. Collectively, our findings uncover a novel mechanism that governs CD73 proteolysis and point to a new therapeutic strategy by modulating CD73 ubiquitylation. Citation Format: Ziyi Fu, Yueming Zhu, Kevin Kalinsky, Yong Wan. Proteolytic regulation of CD73 by TRIM2 orchestrates tumor immunogenicity [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-15.

Proteolytic regulation of CD73 by TRIM21 orchestrates tumor immunogenicity.

Despite the rapid utilization of immunotherapy, emerging challenges to the current immune checkpoint blockade need to be resolved. Here, we report that elevation of CD73 levels due to its aberrant turnover is correlated with poor prognosis in immune-cold triple-negative breast cancers (TNBCs). We have identified TRIM21 as an E3 ligase that governs CD73 destruction. Disruption of TRIM21 stabilizes CD73 that in turn enhances CD73-catalyzed production of adenosine, resulting in the suppression of CD8+ T cell function. Replacement of lysine 133, 208, 262, and 321 residues by arginine on CD73 attenuated CD73 ubiquitylation and degradation. Diminishing of CD73 ubiquitylation remarkably promotes tumor growth and impedes antitumor immunity. In addition, a TRIM21high/CD73low signature in a subgroup of human breast malignancies was associated with a favorable immune profile. Collectively, our findings uncover a mechanism that governs CD73 proteolysis and point to a new therapeutic strategy by modulating CD73 ubiquitylation.

A role for EMT in CD73 regulation in breast cancer

ABSTRACT CD73 is an emerging target in cancer due to its role in generating adenosine, a potent immunosuppressor. We found that SNAI1, a driver of epithelial-to-mesenchymal transition (EMT), upregulates CD73 in triple negative breast cancer cells. Here, we discuss the relevance of improving CD73-based therapy by combining with inhibitors of EMT.

SNAI1-dependent upregulation of CD73 increases extracellular adenosine release to mediate immune suppression in TNBC

Triple-negative subtype of breast cancer (TNBC) is hallmarked by frequent disease relapse and shows highest mortality rate. Although PD-1/PD-L1 immune checkpoint blockades have recently shown promising clinical benefits, the overall response rate remains largely insufficient. Hence, alternative therapeutic approaches are warranted. Given the immunosuppressive properties of CD73-mediated adenosine release, CD73 blocking approaches are emerging as attractive strategies in cancer immunotherapy. Understanding the precise mechanism regulating the expression of CD73 is required to develop effective anti-CD73-based therapy. Our previous observations demonstrate that the transcription factors driving epithelial-to-mesenchymal transition (EMT-TF) can regulate the expression of several inhibitory immune checkpoints. Here we analyzed the role of the EMT-TF SNAI1 in the regulation of CD73 in TNBC cells. We found that doxycycline-driven SNAI1 expression in the epithelial -like TNBC cell line MDA-MB-468 results in CD73 upregulation by direct binding to the CD73 proximal promoter. SNAI1-dependent upregulation of CD73 leads to increased production and release of extracellular adenosine by TNBC cells and contributes to the enhancement of TNBC immunosuppressive properties. Our data are validated in TNBC samples by showing a positive correlation between the mRNA expression of CD73 and SNAI1. Overall, our results reveal a new CD73 regulation mechanism in TNBC that participates in TNBC-mediated immunosuppression and paves the way for developing new treatment opportunities for CD73-positive TNBC.

The regulation of CD73 in non-small cell lung cancer

BackgroundLung cancer is the leading cause of global cancer-related mortality. Although immune checkpoint therapy has achieved remarkable results in lung cancer, EGFR-mutant or ALK-positive non-smallcell lung cancer patients show limited benefit. Besides the low tumor mutational burden, PD-L1 expression and CD8+ tumor-infiltrating T cells, upregulation of CD73/adenosine pathway also contributes to the immune-inert microenvironment of EGFR-mutant NSCLC. However, the detailed mechanism underlying the regulation of CD73 is unclear. MethodsTCGA data was used to analyze the CD73 expression in cancer patients. Western blotting, qPCR, and ChIP-PCR were performed in multiple NSCLC cancer cell lines and patient derived organoids were used to explore the regulation of CD73 expression using western blotting. ResultsCD73 expression was highly expressed in multiple cancer types. Pharmacological or genetic inhibition of EGFR, MEK, KRAS, or ALK dramatically reduced the CD73 mRNA and protein expression in NSCLC cancer cells and patient-derived organoids with EGFR mutation, KRAS mutation or ALK-rearrangement. C-Jun overexpression-induced CD73 mRNA and protein expression. ChIP assay showed that c-Jun bind to CD73 genomic regions. ConclusionsHigher CD73 expression in NSCLC cancer cells and patient-derived organoids with EGFR mutation, KRAS mutation or ALK-rearrangement. Mechanistically, CD73 is regulated by ERK-Jun pathway, wherein c-Jun regulates CD73 expression via binding to CD73 genomic regions.

Abstract P5-06-12: Extracellular adenosine synthesis genes regulated by estrogen signaling are associated with cancer aggressiveness and poor prognosis in estrogen receptor (ER)-positive breast cancer

Abstract Background: Accumulation of extracellular adenosine regulates tumor progression. Extracellular adenosine binds to adenosine receptors, which mediates signaling to induce angiogenesis and cell proliferation, as well as functioning as an immunosuppressive agent in the tumor microenvironment (TME). CD73 and CD39 are two cell surface enzymes that catalyze the synthesis of extracellular adenosine from AMP, ADP and ATP in the TME. However, the underlying mechanisms that regulate adenosine synthesis in the TME of ER-positive breast cancer remains unknown. Methods: In order to investigate the transcriptional regulation of CD73 and CD39 in ER-positive breast cancer, we treated ER-positive breast cancer cell line, MCF7 with estrogen and tamoxifen. We also investigated the clinical significance of CD73/39 expression in ER-positive patients by bioinformatical approach using TCGA and GEO breast cancer cohorts. Results: In TCGA cohort, higher CD73 expression was associated with worse overall survival in ER-positive tumors (p=0.003), but not in ER-negative tumors. Gene set enrichment analysis revealed that estrogen response gene sets (Early; p=0.043, Late; p=0.021) were significantly enriched in CD73 low expressing ER-positive tumors, suggesting estrogen signaling may inhibit CD73 expression. To test this hypothesis, we analyzed the expression of CD73 and CD39 in MCF7 cells treated with estrogen, tamoxifen, or vehicle control. Our data revealed that estrogen treatment suppressed CD73 and CD39 expression, however tamoxifen treatment significantly enhanced expression of the genes. These findings suggest that tamoxifen treatment can induce the expression of CD73 and CD39 in ER-positive breast tumors, by removing the repressive effect of hormone signaling. Additionally, gene set enrichment analysis revealed that CD73-high ER-positive tumor was significantly associated with cancer aggressiveness characteristics, such as epithelial-mesenchymal transition (EMT) (p<0.001) and angiogenesis (p<0.001). On the other hand, CD73-high ER-positive tumor have significantly less infiltrating CD8-positive T cells, memory B cells and plasma cells in silico analysis, implying that CD73-high tumors have immunosuppressive environment. Further, we found that CD73 expression was significantly elevated post-chemotherapy as compared to the tumors prior to the treatment (p=0.007), and CD73 high expressing patients demonstrated worse relapse-free survival in the neoadjuvant chemotherapy cohort (p=0.003). Conclusion: Our molecular findings indicate that expression of CD73 and CD39 are transcriptionally repressed by estrogen signaling, however tamoxifen treatment reverses the effect. Increased expression of CD73 significantly correlates with worse outcomes in ER-positive breast cancer patients which may be due to immunosuppressive tumor microenvironment created by extracellular adenosine driving a pro-metastatic phenotype. Our data indicate an intriguing mechanism which could be therapeutically exploited by targeting CD73/39 to reverse ‘immune-cold’ microenvironment for the treatment of recurrent and metastatic ER-positive breast cancers. Citation Format: Eriko Katsuta, Tao Dai, Abhisha Sawant Dessai, Subhamoy Dasgupta. Extracellular adenosine synthesis genes regulated by estrogen signaling are associated with cancer aggressiveness and poor prognosis in estrogen receptor (ER)-positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-12.

Expression and Regulation of CD73 during the Estrous Cycle in Mouse Uterus.

Cluster of differentiation 73 (CD73, also known as ecto-5′-nucleotidase) is an enzyme that converts AMP into adenosine. CD73 is a surface enzyme bound to the outside of the plasma membrane expressed in several cells and regulates immunity and inflammation. In particular, it is known to inhibit T cell-mediated immune responses. However, the regulation of CD73 expression by hormones in the uterus is not yet clearly known. In this study, we investigated the expression of CD73 in ovariectomized mice treated with estrogen or progesterone and its regulation in the mouse uterus during the estrous cycle. The level of CD73 expression was dynamically regulated in the uterus during the estrous cycle. CD73 protein expression was high in proestrus, estrus, and diestrus, whereas it was relatively low in the metestrus stage. Immunofluorescence revealed that CD73 was predominantly expressed in the cytoplasm of the luminal and glandular epithelium and the stroma of the endometrium. The expression of CD73 in ovariectomized mice was gradually increased by progesterone treatment. However, estrogen injection did not affect its expression. Moreover, CD73 expression was increased when estrogen and progesterone were co-administered and was inhibited by the pretreatment of the progesterone receptor antagonist RU486. These findings suggest that the expression of CD73 is dynamically regulated by estrogen and progesterone in the uterine environment, and that there may be a synergistic effect of estrogen and progesterone.

Identification of the Mouse T Cell ADP-Ribosylome Uncovers ARTC2.2 Mediated Regulation of CD73 by ADP-Ribosylation.

Mouse T cells express the ecto-ADP-ribosyltransferase ARTC2.2, which can transfer the ADP-ribose group of extracellular nicotinamide adenine dinucleotide (NAD+) to arginine residues of various cell surface proteins thereby influencing their function. Several targets of ARTC2.2, such as P2X7, CD8a and CD25 have been identified, however a comprehensive mouse T cell surface ADP-ribosylome analysis is currently missing. Using the Af1521 macrodomain-based enrichment of ADP-ribosylated peptides and mass spectrometry, we identified 93 ADP-ribsoylated peptides corresponding to 67 distinct T cell proteins, including known targets such as CD8a and CD25 but also previously unknown targets such as CD73. We evaluated the impact of ADP-ribosylation on the capability of CD73 to generate adenosine from adenosine monophosphate. Our results show that extracellular NAD+ reduces the enzymatic activity of CD73 HEK cells co-transfected with CD73/ARTC2.2. Importantly, NAD+ significantly reduced CD73 activity on WT CD8 T cells compared to ARTC2ko CD8 T cells or WT CD8 T cells treated with an ARTC2.2-blocking nanobody. Our study provides a comprehensive list of T cell membrane proteins that serve as targets for ADP-ribosylation by ARTC2.2 and whose function may be therefore affected by ADP-ribosylation.

CD73 Is Regulated by the EGFR-ERK Signaling Pathway in Non-small Cell Lung Cancer.

Successful therapy of EGFR-mutant NSCLC remains a challenging task despite initial benefits with the usage of EGFR tyrosine kinase inhibitors. Cancer immunotherapy has shown promising results in certain tumors, but response rate in EGFR-mutant NCLC is low, because these tumors are thought to have weak immunogenicity. We used data from in vivo NSCLC databases as well as from in vitro cell culture experiments to investigate the regulation of CD73 by EGFR. EGFR expression was correlated with CD73 expression in patients' datasets, with EGFR-mutant tumors showing higher expression than their EGFR wildtype counterparts. Treatment of EGFR-mutant NSCLC cell lines with EGFR TKI reduced expression of CD73 at both mRNA and protein level. Among EGFR downstream signaling pathways, the Ras-Raf-ERK pathway was involved in the regulation of CD73 expression directly via ERK1/2 without the engagement of RSKs or MSKs. The results of this study may provide novel therapeutic strategies for the treatment of oncogene-driven NSCLC.

CD73 alleviates GSDMD-mediated microglia pyroptosis in spinal cord injury through PI3K/AKT/Foxo1 signaling.

BackgroundNeuroinflammation‐induced secondary injury is an important cause of sustained progression of spinal cord injury. Inflammatory programmed cell death pyroptosis executed by the pore‐forming protein gasdermin D (GSDMD) is an essential step of neuroinflammation. However, it is unclear whether CD73, a widely accepted immunosuppressive molecule, can inhibit pyroptosis via mediating GSDMD.MethodsC57BL/6J CD73 deficient mice and wild‐type mice, Lipopolysaccharide (LPS)‐induced primary microglia and BV2 cells were respectively used to illustrate the effect of CD73 on microglia pyroptosis in vivo and in vitro. A combination of molecular and histological methods was performed to assess pyroptosis and explore the mechanism both in vivo and in vitro.ResultsWe have shown molecular evidence for CD73 suppresses the activation of NLRP3 inflammasome complexes to reduce the maturation of GSDMD, leading to decreased pyroptosis in microglia. Further analysis reveals that adenosine‐A2B adenosine receptor‐PI3K‐AKT‐Foxo1 cascade is a possible mechanism of CD73 regulation. Importantly, we determine that CD73 inhibits the expression of GSDMD at the transcriptional level through Foxo1. What's more, we confirm the accumulation of HIF‐1α promotes the overexpression of CD73 after spinal cord injury (SCI), and the increased CD73 in turn upregulates the expression of HIF‐1α, eventually forming a positive feedback regulatory loop.ConclusionOur data reveal a novel function of CD73 on microglia pyroptosis, suggesting a unique therapeutic opportunity for mitigating the disease process in SCI.

Cell type- and tissue-specific functions of ecto-5'-nucleotidase (CD73).

Ecto-5'-nucleotidase [cluster of differentiation 73 (CD73)] is a ubiquitously expressed glycosylphosphatidylinositol-anchored glycoprotein that converts extracellular adenosine 5'-monophosphate to adenosine. Anti-CD73 inhibitory antibodies are currently undergoing clinical testing for cancer immunotherapy. However, many protective physiological functions of CD73 need to be taken into account for new targeted therapies. This review examines CD73 functions in multiple organ systems and cell types, with a particular focus on novel findings from the last 5 years. Missense loss-of-function mutations in the CD73-encoding gene NT5E cause the rare disease "arterial calcifications due to deficiency of CD73." Aside from direct human disease involvement, cellular and animal model studies have revealed key functions of CD73 in tissue homeostasis and pathology across multiple organ systems. In the context of the central nervous system, CD73 is antinociceptive and protects against inflammatory damage, while also contributing to age-dependent decline in cortical plasticity. CD73 preserves barrier function in multiple tissues, a role that is most evident in the respiratory system, where it inhibits endothelial permeability in an adenosine-dependent manner. CD73 has important cardioprotective functions during myocardial infarction and heart failure. Under ischemia-reperfusion injury conditions, rapid and sustained induction of CD73 confers protection in the liver and kidney. In some cases, the mechanism by which CD73 mediates tissue injury is less clear. For example, CD73 has a promoting role in liver fibrosis but is protective in lung fibrosis. Future studies that integrate CD73 regulation and function at the cellular level with physiological responses will improve its utility as a disease target.

Tumor-Selective Altered Glycosylation and Functional Attenuation of CD73 in Human Hepatocellular Carcinoma.

CD73, a cell‐surface N‐linked glycoprotein that produces extracellular adenosine, is a novel target for cancer immunotherapy. Although anti‐CD73 antibodies have entered clinical development, CD73 has both protumor and antitumor functions, depending on the target cell and tumor type. The aim of this study was to characterize CD73 regulation in human hepatocellular carcinoma (HCC). We examined CD73 expression, localization, and activity using molecular, biochemical, and cellular analyses on primary HCC surgical specimens, coupled with mechanistic studies in HCC cells. We analyzed CD73 glycan signatures and global alterations in transcripts encoding other N‐linked glycoproteins by using mass spectrometry glycomics and RNA sequencing (RNAseq), respectively. CD73 was expressed on tumor hepatocytes where it exhibited abnormal N‐linked glycosylation, independent of HCC etiology, tumor stage, or fibrosis presence. Aberrant glycosylation of tumor‐associated CD73 resulted in a 3‐fold decrease in 5′‐nucleotidase activity (P < 0.0001). Biochemically, tumor‐associated CD73 was deficient in hybrid and complex glycans specifically on residues N311 and N333 located in the C‐terminal catalytic domain. Blocking N311/N333 glycosylation by site‐directed mutagenesis produced CD73 with significantly decreased 5′‐nucleotidase activity in vitro, similar to the primary tumors. Glycosylation‐deficient CD73 partially colocalized with the Golgi structural protein GM130, which was strongly induced in HCC tumors. RNAseq analysis further revealed that N‐linked glycoprotein‐encoding genes represented the largest category of differentially expressed genes between HCC tumor and adjacent tissue. Conclusion: We provide the first detailed characterization of CD73 glycosylation in normal and tumor tissue, revealing a novel mechanism that leads to the functional suppression of CD73 in human HCC tumor cells. The present findings have translational implications for therapeutic candidate antibodies targeting cell‐surface CD73 in solid tumors and small‐molecule adenosine receptor agonists that are in clinical development for HCC.

The distinct role of CD73 in the progression of pancreatic cancer

Recent studies have shown that the non-enzymatic function of CD73 plays a key role in tumor progression, but this function of CD73 in pancreatic cancer cells has not been studied. Furthermore, little is known about the mechanism involved in CD73 regulation in tumors. Here, we found that CD73 expression was upregulated in pancreatic ductal adenocarcinoma (PDAC) and that its expression correlated with poor prognosis. CD73 knockdown inhibited cell growth and induced G1 phase arrest via the AKT/ERK/cyclin D signaling pathway. We also found that tumor necrosis factor receptor (TNFR) 2 was involved in CD73-induced AKT and ERK signaling pathway activation in PDAC. Further, miR-30a-5p overexpression significantly increased the cytotoxic effect of gemcitabine in pancreatic cancer by directly targeting CD73 messenger RNA (mRNA), suggesting that regulation of the miR-30a-5p/CD73 axis may play an important role in the development of gemcitabine resistance in pancreatic cancer. In summary, this regulatory network of CD73 appears to represent a new molecular mechanism underlying PDAC progression, and the mechanistic interaction between miR-30a-5p, CD73, and TNFR2 may provide new insights into therapeutic strategies for pancreatic cancer.Key messagesCD73 was upregulated in PDAC and correlated with poor prognosis.CD73 knockdown inhibited cell growth and induced G1 phase arrest.TNFR2 was involved in CD73-induced AKT and ERK signaling pathway.miR-30a-5p targeted CD73 and increased the sensitivity to gemcitabine.

CD73 Alleviates GSDMD-Mediated Pyroptosis in Spinal Cord Injury Through PI3K/AKT/Foxo1 Signaling

Neuroinflammation-induced secondary injury is an important cause of sustained progression of spinal cord injury. Inflammatory programmed cell death pyroptosis executed by the pore-forming protein gasdermin D (GSDMD) is recently regarded as an essential step of neuroinflammation. However, it is unclear whether CD73, a widely accepted immunosuppressive molecule, can inhibit pyroptosis via mediating GSDMD. Here, we reported molecular evidence for CD73 suppresses the activation of NLRP3 inflammasome complexes to reduce the maturation of GSDMD, leading to decreased pyroptosis in macrophages/microglia. Further analysis revealed that adenosine-A2B adenosine receptor-NF-κB cascade is a possible mechanism of CD73 regulation. Importantly, we determined that CD73 inhibited the expression of GSDMD at the transcriptional level through NF-κB, thereby alleviating macrophages/microglia pyroptosis. What’s more, we confirmed the accumulation of HIF-1α promotes the overexpression of CD73 after SCI, and the increased CD73 in turn upregulates the expression of HIF-1α, eventually forming a positive feedback regulatory loop. In summary, our results demonstrate that CD73 alleviates GSDMD-mediated pyroptosis of macrophages/microglia in spinal cord injury by blocking NF-κB signal. Our data reveal a novel function of CD73 on microphages/microglia pyroptosis, suggesting a unique therapeutic opportunity for mitigating the disease process in SCI. Funding Statement: This work was supported by the National Natural Science Foundation of China [grant number 81772385, 81871522]. Declaration of Interests: The authors declare that they do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Ethics Approval Statement: All study surgical procedures and experiment protocols were performed in accordance with standard guidelines approved by the Ethics Committee of Experimental Research, Shanghai Medical College, Fudan University (Shanghai, China). The animal experiments were approved by the Institutional Animal Care and Use Committee of Fudan University.

The immunosuppressive effect of the tick protein, Salp15, is long-lasting and persists in a murine model of hematopoietic transplant

Salp15, a salivary protein of Ixodes ticks, inhibits the activation of naïve CD4 T cells. Treatment with Salp15 results in the inhibition of early signaling events and the production of the autocrine growth factor, interleukin-2. The fate of the CD4 T cells activated in the presence of Salp15 or its long-term effects are, however, unknown. We now show that Salp15 binding to CD4 is persistent and induces a long-lasting immunomodulatory effect. The activity of Salp15 results in sustained diminished cross-antigenic antibody production even after interruption of the treatment with the protein. Transcriptionally, the salivary protein provokes an acute effect that includes known activation markers, such as Il2 or Cd44, and that fades over time. The long-term effects exerted by Salp15 do not involve the induction of either anergy traits nor increased populations of regulatory T cells. Similarly, the treatment with Salp15 does not result in B cell anergy or the generation of myeloid suppressor cells. However, Salp15 induces the increased expression of the ectoenzyme, CD73, in regulatory T cells and increased production of adenosine. Our study provides a profound characterization of the immunomodulatory activity of Salp15 and suggests that its long-term effects are due to the specific regulation of CD73.

Expression of CD73 slows down migration of skin dendritic cells, affecting the sensitization phase of contact hypersensitivity reactions in mice

BackgroundApplication of haptens to the skin induces release of immune stimulatory ATP into the extracellular space. This “danger” signal can be converted to immunosuppressive adenosine (ADO) by the action of the ectonucleotidases CD39 and CD73, expressed by skin and immune cells. Thus, the expression and regulation of CD73 by skin derived cells may have crucial influence on the outcome of contact hypersensitivity (CHS) reactions. ObjectiveTo investigate the role of CD73 expression during 2,4,6-trinitrochlorobenzene (TNCB) induced CHS reactions. MethodsWild type (wt) and CD73 deficient mice were subjected to TNCB induced CHS. In the different mouse strains the resulting ear swelling reaction was recorded along with a detailed phenotypic analysis of the skin migrating subsets of dendritic cells (DC). ResultsIn CD73 deficient animals the motility of DC was higher as compared to wt animals and in particular after sensitization we found increased migration of Langerin+ DC from skin to draining lymph nodes (LN). In the TNCB model this led to a stronger sensitization as indicated by increased frequency of interferon-γ producing T cells in the LN and an increased ear thickness after challenge. ConclusionCD73 derived ADO production slows down migration of Langerin+ DC from skin to LN. This may be a crucial mechanism to avoid over boarding immune reactions against haptens.

CD73/NT5E is a target of miR-30a-5p and plays an important role in the pathogenesis of non-small cell lung cancer

BackgroundCD73 (ecto-5′-nucleotidase) is implicated in the development of many types of cancer. CD73 inhibitors are currently being tested in clinical trials for the treatment of cancer. Understanding the molecular and cellular actions of CD73 inhibitors is the key to improving this line of therapy.MethodsQuantitative real-time PCR (qRT-PCR) was used to detect the expression of CD73 and miR-30a-5p; Western blot and immunohistochemical assays were used to investigate the levels of CD73 and other proteins. Flow cytometry was used to determine cell cycle stage and apoptosis. CCK-8 and clonogenic assays were used to investigate cell proliferation. Wound healing, migration and invasion assays were used to investigate the motility of cells. A lung carcinoma xenograft mouse model was used to investigate the in vivo effects of CD73 and miR-30a-5p.ResultsIn the present study, we found that CD73 is overexpressed and miR-30a-5p is underexpressed in non-small cell lung cancer tissues compared with adjacent noncancerous. Further, we showed that CD73 is a direct target of miR-30a-5p by luciferase reporter assays, qRT-PCR and western blot analysis. We also found that overexpression of miR-30a-5p in these non-small cell lung cancer cell lines inhibited cell proliferation in vitro and in vivo. Moreover, the epithelial-to-mesenchymal phenotype was suppressed and cell migration and invasion were inhibited; these effects were brought about via the EGF signaling pathway.ConclusionsOur findings reveal a new post-transcriptional mechanism of CD73 regulation via miR-30a-5p and EGFR-related drug resistance in non-small cell lung cancer.

Regulation of CD73 in the development of lower limb atherosclerosis.

Atherosclerosis is an inflammatory process of the arterial wall. CD73 (also known as ecto-5'-nucleotidase) is a key regulator of cell signaling in response to inflammation and hypoxia, and may be important in the development of atherosclerosis. Recently, we have shown that high CD73 activity can be detected in the serum of patients with peripheral arterial disease (PAD). Using this same PAD patient cohort of 226 subjects with 38 femoral artery samples obtained during surgical endarcterectomy and control artery samples taken during autopsy, we explored the association of serum CD73 activity with overall atherosclerotic burden and the expression of CD73 in mature and developing plaques. Interestingly, we found that CD73 activity had a tendency to increase along with more severe presentation of PAD (from 249nmol/mL/h in moderate disease to 332nmol/mL/h in severe disease; P=0.013) and that CD73 expression is elevated in the vasa vasorum of developing plaques, but completely lost in mature occlusive plaques removed during endarcterectomy (P<0.001). The current findings implicate that as a result of shedding and loss of CD73 from the arterial wall, CD73 activity is elevated in the serum of patients with widespread atherosclerosis. These findings highlight the importance of a better understanding of the local role of CD73 in the development and maturation of arterial atherosclerotic plaques in man.