apyrase and Lung-Neoplasms

An important mechanism, by which cancer cells achieve immune escape, is the release of extracellular adenosine into their microenvironment. Adenosine activates adenosine A. We pursued a repurposing approach by screening a self-compiled collection of approved, mostly ATP-competitive protein kinase inhibitors, on human CD39. The best hit compound was further characterized and evaluated in various orthogonal assays and enzyme preparations, and on human immune and cancer cells.. The tyrosine kinase inhibitor ceritinib, a potent anticancer drug used for the treatment of anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer, was found to strongly inhibit CD39 showing selectivity versus other ectonucleotidases. The drug displays a non-competitive, allosteric mechanism of CD39 inhibition exhibiting potency in the low micromolar range, which is independent of substrate (ATP) concentration. We could show that ceritinib inhibits ATP dephosphorylation in peripheral blood mononuclear cells in a dose-dependent manner, resulting in a significant increase in ATP concentrations and preventing adenosine formation from ATP. Importantly, ceritinib (1-10 µM) substantially inhibited ATP hydrolysis in triple negative breast cancer and melanoma cells with high native expression of CD39.. CD39 inhibition might contribute to the effects of the powerful anticancer drug ceritinib. Ceritinib is a novel CD39 inhibitor with high metabolic stability and optimized physicochemical properties; according to our knowledge, it is the first brain-permeant CD39 inhibitor. Our discovery will provide the basis (i) to develop more potent and balanced dual CD39/ALK inhibitors, and (ii) to optimize the ceritinib scaffold towards interaction with CD39 to obtain potent and selective drug-like CD39 inhibitors for future in vivo studies.

Topics: Adenosine; Adenosine Triphosphate; Antigens, CD; Antineoplastic Agents; Apyrase; Carcinoma, Non-Small-Cell Lung; Humans; Immunotherapy; Leukocytes, Mononuclear; Lung Neoplasms; Protein Kinase Inhibitors; Pyrimidines; Receptor Protein-Tyrosine Kinases; Sulfones; Tumor Microenvironment

Immune related endonucleases have recently been described as potential therapeutic targets and predictors of response to treatment with immune checkpoint inhibitors (ICI). The aim is to evaluate the association between the expression of 5 biomarkers involved in the immune response (CD73, CD39, VISTA, Arl4d and Cytohesin-3) in parallel with the more common ICI-predictive markers, PD-L1 expression and Tumor Mutation Burden (TMB) with response to ICI therapy in an advanced non-small cell lung cancer (NSCLC) cohort.. Patients with advanced NSCLC treated with ICI single agent were divided into responders and non-responders according to RECIST v1.1 and duration of response (DOR) criteria. Immunohistochemistry was performed on pretreatment tumor tissue samples for PD-L1, CD73, CD39, VISTA, Arl4d, and Cytohesin-3 expression. TMB was estimated with NEOplus v2 RUO (NEO New Oncology GmbH) hybrid capture next generation sequencing assay. Resistance mutations in STK11/KEAP1 and positive predictive mutations in ARID1A/POLE were also evaluated.. Included were 56 patients who were treated with ICI single agent. The median progression-free and overall survival for the whole cohort was 3.0 (95% CI, 2.4-3.6) and 15 (95% CI, 9.7-20.2) months, respectively. The distribution of CD73 in tumor cells and CD39, VISTA, Arl4d and Cytohesin-3 expression in immune cells were not different between responders and non-responders. Also, PD-L1 and TMB were not predictive for response. The frequency of STK11, KEAP1 and ARID1A mutations was low and only observed in the non-responder group.. Separate and combined expression of 5 biomarkers involved in the immune response (CD73, CD39, VISTA, Arl4d, and Cytohesin-3) was not associated with response in our cohort of advanced NSCLC patients receiving single agent ICI. To confirm our findings the analysis of independent larger cohorts is warranted.

Topics: 5'-Nucleotidase; ADP-Ribosylation Factors; Aged; Aged, 80 and over; Antibodies, Monoclonal, Humanized; Apyrase; B7 Antigens; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Disease Progression; Female; GPI-Linked Proteins; Humans; Immune Checkpoint Inhibitors; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Mutation; Nivolumab; Predictive Value of Tests; Progression-Free Survival; Receptors, Cytoplasmic and Nuclear; Time Factors

Natural killer (NK) cell protection from tumor metastases is a critical feature of the host immune response to cancer, but various immunosuppression mechanisms limit NK cell effector function. The ectoenzyme, CD39, expressed on tumor-infiltrating myeloid cells, granulocytes, and lymphocytes, including NK cells, converts extracellular ATP (eATP) into AMP and, thus, potentially suppresses eATP-mediated proinflammatory responses. A CD39-targeting monoclonal antibody (mAb) that inhibits the mouse ectoenzyme CD39 suppressed experimental and spontaneous metastases in a number of different tumor models and displayed superior antimetastatic activity compared with the CD39 inhibitor POM1 and inhibitors and mAbs that block other members of the adenosinergic family (e.g., A2AR and CD73). The antimetastatic activity of anti-CD39 was NK cell and IFNγ dependent, and anti-CD39 enhanced the percentage and quantity of IFNγ produced and CD107a expression in lung-infiltrating NK cells following tumor challenge and anti-CD39 therapy. Using conditional

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Apyrase; Immune Tolerance; Inflammasomes; Kidney Neoplasms; Killer Cells, Natural; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Myeloid Cells; Tumor Cells, Cultured

The major suppressive immune cells in tumor sites are myeloid derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and Treg cells, and the major roles of these suppressive immune cells include hindering T-cell activities and supporting tumor progression and survival. In this study, we analyzed the pattern of circulating MDSC subtypes in patients with non-small cell lung cancer (NSCLC) whether those suppressive immune cells hinder T-cell activities leading to poor clinical outcomes. First, we verified PMN-MDSCs, monocytic-MDSCs (M-MDSCs), and Treg cells increased according to the stages of NSCLC, and MDSCs effectively suppressed T-cell activities and induced T-cell exhaustion. The analysis of NSCLC patients treated with anti-PD-1 immunotherapy demonstrated that low PMN-MDSCs, M-MDSCs, and CD39

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Apyrase; Carcinoma, Non-Small-Cell Lung; CD8-Positive T-Lymphocytes; Female; Humans; Immunotherapy; Lung Neoplasms; Lymphocyte Activation; Male; Middle Aged; Myeloid-Derived Suppressor Cells; Programmed Cell Death 1 Receptor

Adenosine triphosphate (ATP) is released at a high concentration in the tumor microenvironment. The overexpression of ectonucleotidases in non-small-cell lung cancer (NSCLC), metabolizing ΑΤP to the immunosuppressive adenosine, is studied.. We examined the expression of the ectonucleotidases CD73 and CD39 in NSCLC in parallel with immunological parameters and markers of hypoxia and anaerobic metabolism. In vitro experiments with A549 and H1299 lung cancer cell lines were also conducted.. CD73 and CD39 were not expressed by normal bronchial and alveolar epithelium. In contrast, these were overexpressed by cancer cells, cancer-associated fibroblasts (CAFs), and tumor-infiltrating lymphocytes (TILs). High CD73 cancer cell expression was directly linked with lactate dehydrogenase LDH5 and with hypoxia-inducible factor HIF1α expression by cancer cells. The expression of CD39 by CAFs was directly linked with PD-L1 expression by cancer cells. A significant abundance of FOXP3+ and PD-1+ TILs was noted in tumors with high CD73 and CD39 stroma expression. In in vitro experiments, hypoxia and acidity induced CD73 mRNA and protein levels in cancer cell lines. Exposure of cancer cell lines to adenosine induced the expression of PD-L1 and LDHA mRNA and protein levels.. Ectonucleotidases are up-regulated in cancer cells, CAFs, and TILs in lung tumors. Such overexpression is linked with regulatory TIL-phenotype and PD-L1 up-regulation by cancer cells. Overexpression of LDH5 is up-regulated by adenosine, creating a vicious cycle, as the high amounts of ATP produced by LDH5-mediated anaerobic glycolysis promote the production of adenosine by a tumor microenvironment rich in ectonucleotidases.

Topics: 5'-Nucleotidase; Adenosine; Adult; Aged; Aged, 80 and over; Antigens, CD; Apyrase; B7-H1 Antigen; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Immune Tolerance; Lung Neoplasms; Male; Middle Aged

The aim of this study was to investigate the effects and mechanisms of ectonucleoside triphosphate phosphohydrolase-7 (ENTPD7) on lung cancer cells.. The expression characteristics of ENTPD7 and its effect on the survival of lung cancer patients were analyzed by referring to The Cancer Genome Atlas (TCGA). Streptavidin-peroxidase (SP) staining was performed to detect the ENTPD7 protein in tumor tissues and adjacent tissues. Plasmid transfection technology was also applied to silence ENTPD7 gene. Crystal violet staining and flow cytometry were performed to determine cell proliferation and apoptosis. Tumor-bearing nude mice model was established to investigate the effect of sh-ENTPD7 on tumors.. The results showed that patients with low levels of ENTPD7 had higher survival rates. ENTPD7 was up-regulated in lung cancer tissues and cells. Down-regulation of the expression of ENTPD7 inhibited proliferation but promoted apoptosis of lung cancer cell. Silencing ENTPD7 also inhibited the expression levels of Ras and Raf proteins and the phosphorylation of mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK). Tumor-bearing nude mice experiments showed that silencing ENTPD7 had an inhibitory effect on lung cancer cells.. ENTPD7 was overexpressed in lung cancer cells. Down-regulating ENTPD7 could inhibit lung cancer cell proliferation and promote apoptosis via inhibiting the Ras/Raf/MEK/ERK pathway.

Topics: Adult; Aged; Animals; Apoptosis; Apyrase; Biomarkers; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Lung Neoplasms; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Mitogen-Activated Protein Kinases; Plasmids; raf Kinases; ras Proteins; Signal Transduction; Survival Analysis

Various forms of immunotherapy, such as checkpoint blockade immunotherapy, are proving to be effective at restoring T cell-mediated immune responses that can lead to marked and sustained clinical responses, but only in some patients and cancer types

Topics: Antigens, Neoplasm; Antigens, Viral; Apyrase; Bystander Effect; CD8-Positive T-Lymphocytes; Cell Separation; Colorectal Neoplasms; ErbB Receptors; Humans; Lung Neoplasms; Lymphocytes, Tumor-Infiltrating; Phenotype

Purines are well-known as intracellular sources for energy but they also act as extracellular signaling molecules. In the recent years, there has been a growing interest in the therapeutic potential of purinergic signaling for cancer treatment. This is the first study to analyze lung purine levels and purinergic receptors in non-small-cell lung cancer (NSCLC) patients.. In this prospective clinical trial we enrolled 26 patients with NSCLC and 21 patients with chronic obstructive pulmonary disease (COPD) without signs of malignancy. The purine concentrations were analyzed in bronchoalveolar lavage fluid (BALF) using fluorescent/luminescent assays. Expression of purinergic receptors and ectonucleotidases were analyzed using real time quantitative polymerase chain reaction (RT-qPCR).. Patients with NSCLC have significantly lower ATP and ADP concentrations in BALF than patients with COPD (p=0.006 and p=0.009). Expression of the ectonucleotidase CD39 is significantly higher in BAL cells from cancer patients compared to COPD (p=0.001) as well as in metastasized tumors compared to non-metastasized tumors (p=0.009). Receptor-analysis revealed a higher expression of P2X4 (p=0.03), P2X7 (p=0.001) and P2Y1 (p=0.003) in BAL cells of tumors with distant metastasis.. Our data suggests a role for CD39 in lung cancer tumor microenvironment, influencing tumor invasiveness and metastasization. Potentially the increased degradation of ATP and ADP leads to a subversion of their anti-neoplastic effects. Furthermore P2Y1, P2X4 and P2X7 receptors are upregulated in BAL cells in metastatic disease. Our findings might facilitate the identification of new therapeutic targets for cancer immunotherapy.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Aged; Antigens, CD; Apyrase; Bronchoalveolar Lavage Fluid; Carcinoma, Non-Small-Cell Lung; Female; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Pulmonary Disease, Chronic Obstructive; Receptors, Purinergic; Signal Transduction; Tumor Microenvironment

To detect the expression levels of co-inhibitory molecules, including CTLA-4, LAG-3, PD-1 and CD39, on CD4⁺ T cells in peripheral blood or tumor tissues from NSCLC patients and to investigate their potential internal relationships with the progression of NSCLC.. Eighty-eight patients including 53 NSCLC, 17 disease control cases and 18 healthy controls were studied. All the peripheral blood and 13 cases of tumor and tumor-adjacent tissues from surgically treated NSCLC patients were obtained. The expression levels of co-inhibitory molecules CTLA-4, LAG-3, PD-1 and CD39 were assayed by flow cytometry (FCM).. The ratios of CD4⁺ CTLA-4⁺ T cells, CD4⁺ LAG-3⁺ T cells, CD4⁺ PD-1⁺ T cells and CD4⁺ CD39⁺ T cells in the peripheral blood of NSCLC patients were (2.49 ± 2.43)%, (2.47 ± 3.50)%, (12.94 ± 5.96)% and (6.78 ± 5.21)%, respectively, the ratio of CD4⁺ CTLA-4⁺ T cells was significantly higher in the peripheral blood of NSCLC patients than that in the disease controls and healthy controls (P < 0.05) . The ratio of CD4(+)PD-1⁺ T cells was also highly raised in the peripheral blood of NSCLC patients than that in the healthy controls (P < 0.05). Further stratified analysis indicated that the ratio of CD4⁺ PD-1⁺ T cells was (13.21 ± 5.96)% in NSCLC patients entering stages III and IV, also significantly increased as compared with that of (11.06 ± 3.42)% in the patients undergoing stages I and II (P < 0.05). More CD4⁺ CTLA-4⁺ T cells, CD4⁺ LAG-3⁺ T cells and CD4⁺ PD-1⁺ T cells were verified in the cancer tissues (5.07 ± 2.11)%, (7.86 ± 3.24)% and (40.20 ± 18.84)%, respectively, than those in their matched peripheral blood (3.13 ± 1.01)%, (2.65 ± 1.48)% and (15.79 ± 5.69)%, (P < 0.05 for all), and especially, CD4⁺ CTLA-4⁺ T cells and CD4⁺ PD-1⁺ T cells were also highly increased than those in matched cancer-adjacent tissues (P < 0.05 for all).. The increased expression levels of co-inhibitory molecules CTLA-4, LAG-3 and PD-1 on CD4⁺ T cells in peripheral blood and tumor tissues may be one of the mechanisms related to immune escape of tumor cells, acceleration of disease progression and poor prognosis in NSCLC patients.

Topics: Antigens, CD; Apyrase; Carcinoma, Non-Small-Cell Lung; CD4-Positive T-Lymphocytes; CTLA-4 Antigen; Disease Progression; Flow Cytometry; Humans; Lung Neoplasms; Programmed Cell Death 1 Receptor

Both regulatory T cells (Tregs) and T helper IL-17-producing cells (Th17 cells) have been found to be involved in human malignancies, however, the possible implication of Tregs in regulating generation and differentiation of Th17 cells in malignant pleural effusion remains to be elucidated.. The numbers of both CD39(+)Tregs and Th17 cells in malignant pleural effusion and peripheral blood from patients with lung cancer were determined by flow cytometry. The regulation and mechanism of Tregs on generation and differentiation of Th17 cells were explored.. Both CD39(+)Tregs and Th17 cells were increased in malignant pleural effusion when compared with blood, and the numbers of CD39(+)Tregs were correlated negatively with those of Th17 cells. It was also noted that high levels of IL-1β, IL-6, and TGF-β1 could be observed in malignant pleural effusion when compared the corresponding serum, and that pleural CD39(+)Tregs could express latency-associated peptide on their surface. When naïve CD4(+) T cells were cocultured with CD39(+)Tregs, Th17 cell numbers decreased as CD39(+)Treg numbers increased, addition of the anti-latency-associated peptide mAb to the coculture reverted the inhibitory effect exerted by CD39(+)Tregs.. Therefore, the above results indicate that CD39(+)Tregs inhibit generation and differentiation of Th17 cells via a latency-associated peptide-dependent mechanism.

Topics: Adult; Aged; Antigens, CD; Apyrase; Cell Differentiation; Cell Proliferation; Cells, Cultured; Coculture Techniques; Flow Cytometry; Humans; Immunophenotyping; Interleukin-1beta; Interleukin-6; Lung Neoplasms; Middle Aged; Peptides; Pleural Effusion, Malignant; Protein Precursors; Signal Transduction; T-Lymphocytes, Regulatory; Th17 Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1

Extracellular ATP is a potent surfactant secretagogue but its origin in the alveolus, its mechanism(s) of release and its regulatory pathways remain unknown. Previously, we showed that hypotonic swelling of alveolar A549 cells induces Ca(2+)-dependent secretion of several adenosine and uridine nucleotides, implicating regulated exocytosis. In this study, we examined sources of Ca(2+) for the elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) evoked by acute 50% hypotonic stress and the role of autocrine purinergic signalling in Ca(2+)-dependent ATP release. We found that ATP release does not directly involve Ca(2+) influx from extracellular spaces, but depends entirely on Ca(2+) mobilization from intracellular stores. The [Ca(2+)](i) response consisted of slowly rising elevation, representing mobilization from thapsigargin (TG)-insensitive stores and a superimposed rapid spike due to Ca(2+) release from TG-sensitive endoplasmic reticulum (ER) Ca(2+) stores. The latter could be abolished by hydrolysis of extracellular triphospho- and diphosphonucleotides with apyrase; blocking P2Y(2)/P2Y(6) receptors of A549 cells with suramin; blocking UDP receptors (P2Y(6)) with pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS); emptying TG-sensitive stores downstream with TG or caffeine in Ca(2+)-free extracellular solution; or blocking the Ca(2+)-release inositol 1,4,5-triphosphate receptor channel of the ER with 2-aminoethyldiphenylborinate. These data demonstrate that the rapid [Ca(2+)](i) spike results from the autocrine stimulation of IP(3)/Ca(2+)-coupled P2Y, predominantly P2Y(6), receptors, accounting for approximately 70% of total Ca(2+)-dependent ATP release evoked by hypotonic shock. Our study reveals a novel paradigm in which stress-induced ATP release from alveolar cells is amplified by the synergistic autocrine/paracrine action of coreleased uridine and adenosine nucleotides. We suggest that a similar mechanism of purinergic signal propagation operates in other cell types.

Topics: Adenosine Triphosphate; Apyrase; Autocrine Communication; Caffeine; Calcium; Calcium Signaling; Calcium-Transporting ATPases; Cell Line, Tumor; Endoplasmic Reticulum; Enzyme Inhibitors; Epithelial Cells; Humans; Hydrolysis; Hypotonic Solutions; Inositol 1,4,5-Trisphosphate Receptors; Lung Neoplasms; Osmotic Pressure; Paracrine Communication; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; Suramin; Thapsigargin; Time Factors; Uracil Nucleotides

We established five clones (ML-01, ML-02, MH-01, MH-02, MH-03) from murine 3-methylcholanthrene-induced fibrosarcoma A (Meth A), and investigated their experimental metastatic potentials in relation to their platelet-aggregating activities. A clone with a high metastatic potential (MH-02) showed a characteristic biphasic pattern of platelet aggregation, of which the first peak was not present in the aggregation patterns of the clone with low metastatic potential (ML-01). The first peak was eliminated by treatment of the cells with apyrase, indicating that adenosine diphosphate (ADP) was the causative substance of this particular peak. The metastatic potential of clones correlated well with the ADP concentration of the culture media. These results suggest that the increased ADP production and consequential enhancement of platelet-aggregating activity are closely related to the increment of pulmonary metastatic potential of MH-02 clone.

Topics: Animals; Apyrase; Fibrosarcoma; Hot Temperature; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Microbial Collagenase; NADP; Neoplasm Metastasis; Neuraminidase; Platelet Aggregation; Trypsin; Tumor Cells, Cultured; Ultracentrifugation

Topics: Adenocarcinoma; Animals; Apyrase; Cell Line; Colonic Neoplasms; Glioma; Hirudins; Humans; Kinetics; Lung Neoplasms; Melanoma; Mesothelioma; Mice; Neoplasms; Neoplasms, Experimental; Neuroblastoma; Phospholipases; Phosphoric Monoester Hydrolases; Platelet Aggregation