Cytosolic 5′-nucleotidase II silencing in a human lung carcinoma cell line opposes cancer phenotype with a concomitant increase in p53 phosphorylation

Purine homeostasis is maintained by a purine cycle in which the regulated member is a cytosolic 5′-nucleotidase II (cN-II) hydrolyzing IMP and GMP. Its expression is particularly high in proliferating cells, indeed high cN-II activity or expression in hematological malignancy has been associated to poor prognosis and chemoresistance. Therefore, a strong interest has grown in developing cN-II inhibitors, as potential drugs alone or in combination with other compounds. As a model to study the effect of cN-II inhibition we utilized a lung carcinoma cell line (A549) in which the enzyme was partially silenced and its low activity conformation was stabilized through incubation with 2-deoxyglucose. We measured nucleotide content, reduced glutathione, activities of enzymes involved in glycolysis and Krebs cycle, protein synthesis, mitochondrial function, cellular proliferation, migration and viability. Our results demonstrate that high cN-II expression is associated with a glycolytic, highly proliferating phenotype, while silencing causes a reduction of proliferation, protein synthesis and migration ability, and an increase of oxidative performances. Similar results were obtained in a human astrocytoma cell line. Moreover, we demonstrate that cN-II silencing is concomitant with p53 phosphorylation, suggesting a possible involvement of this pathway in mediating some of cN-II roles in cancer cell biology

Cytosolic 5'-Nucleotidase II Is a Sensor of Energy Charge and Oxidative Stress: A Possible Function as Metabolic Regulator

Cytosolic 5'-nucleotidase II (NT5C2) is a highly regulated enzyme involved in the maintenance of intracellular purine and the pyrimidine compound pool. It dephosphorylates mainly IMP and GMP but is also active on AMP. This enzyme is highly expressed in tumors, and its activity correlates with a high rate of proliferation. In this paper, we show that the recombinant purified NT5C2, in the presence of a physiological concentration of the inhibitor inorganic phosphate, is very sensitive to changes in the adenylate energy charge, especially from 0.4 to 0.9. The enzyme appears to be very sensitive to pro-oxidant conditions; in this regard, the possible involvement of a disulphide bridge (C175-C547) was investigated by using a C547A mutant NT5C2. Two cultured cell models were used to further assess the sensitivity of the enzyme to oxidative stress conditions. NT5C2, differently from other enzyme activities, was inactivated and not rescued by dithiothreitol in a astrocytoma cell line (ADF) incubated with hydrogen peroxide. The incubation of a human lung carcinoma cell line (A549) with 2-deoxyglucose lowered the cell energy charge and impaired the interaction of NT5C2 with the ice protease-activating factor (IPAF), a protein involved in innate immunity and inflammation

Intracellular Cytidine Deaminase Regulates Gemcitabine Metabolism in Pancreatic Cancer Cell Lines

Cytidine deaminase (CDA) is a determinant of in vivo gemcitabine elimination kinetics and cellular toxicity. The impact of CDA activity in pancreatic ductal adenocarcinoma (PDAC) cell lines has not been elucidated. We hypothesized that CDA regulates gemcitabine flux through its inactivation and activation pathways in PDAC cell lines. Three PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) were incubated with 10 or 100 µM gemcitabine for 60 minutes or 24 hours, with or without tetrahydrouridine, a CDA inhibitor. Extracellular inactive gemcitabine metabolite (dFdU) and intracellular active metabolite (dFdCTP) were quantified with liquid chromatography tandem mass spectrometry. Cellular expression of CDA was assessed with real-time PCR and Western blot. Gemcitabine conversion to dFdU was extensive in BxPC-3 and low in MIA PaCa-2 and PANC-1, in accordance with their respective CDA expression levels. CDA inhibition was associated with low or undetectable dFdU in all three cell lines. After 24 hours gemcitabine incubation, dFdCTP was highest in MIA PaCa-2 and lowest in BxPC-3. CDA inhibition resulted in a profound dFdCTP increase in BxPC-3 but not in MIA PaCa-2 or PANC-1. dFdCTP concentrations were not higher after exposure to 100 versus 10 µM gemcitabine when CDA activities were low (MIA PaCa-2 and PANC-1) or inhibited (BxPC-3). The results suggest a regulatory role of CDA for gemcitabine activation in PDAC cells but within limits related to the capacity in the activation pathway in the cell lines. SIGNIFICANCE STATEMENT The importance of cytidine deaminase (CDA) for cellular gemcitabine toxicity, linking a lower activity to higher toxicity, is well described. An underlying assumption is that CDA, by inactivating gemcitabine, limits the amount available for the intracellular activation pathway. Our study is the first to illustrate this regulatory role of CDA in pancreatic ductal adenocarcinoma cell lines by quantifying intracellular and extracellular gemcitabine metabolite concentrations.publishedVersio

The Antitumor Activity of Combinations of Cytotoxic Chemotherapy and Immune Checkpoint Inhibitors Is Model-Dependent

In spite of impressive response rates in multiple cancer types, immune checkpoint inhibitors (ICIs) are active in only a minority of patients. Alternative strategies currently aim to combine immunotherapies with conventional agents such as cytotoxic chemotherapies. Here, we performed a study of PD-1 or PDL-1 blockade in combination with reference chemotherapies in four fully immunocompetent mouse models of cancer. We analyzed both the in vivo antitumor response, and the tumor immune infiltrate 4 days after the first treatment. in vivo tumor growth experiments revealed variable responsiveness to ICIs between models. We observed enhanced antitumor effects of the combination of immunotherapy with chemotherapy in the MC38 colon and MB49 bladder models, a lack of response in the 4T1 breast model, and an inhibition of ICIs activity in the MBT-2 bladder model. Flow cytometry analysis of tumor samples showed significant differences in all models between untreated and treated mice. At baseline, all the tumor models studied were predominantly infiltrated with cells harboring an immunosuppressive phenotype. Early alterations of the tumor immune infiltrate after treatment were found to be highly variable. We found that the balance between effector cells and immunosuppressive cells in the tumor microenvironment could be altered with some treatment combinations, but this effect was not always correlated with an impact on in vivo tumor growth. These results show that the combination of cytotoxic chemotherapy with ICIs may result in enhanced, similar or reduced antitumor activity, in a model- and regimen-dependent fashion. The present investigations should help to select appropriate combination regimens for ICIs

PARP Inhibitors and Proteins Interacting with SLX4

PARP inhibitors are small molecules currently used with success in the treatment of certain cancer patients. Their action was first shown to be specific to cells with DNA repair deficiencies, such as BRCA-mutant cancers. However, recent work has suggested clinical interest of these drugs beyond this group of patients. Preclinical data on relationships between the activity of PARP inhibitors and other proteins involved in DNA repair exist, and this review will only highlight findings on the SLX4 protein and its interacting protein partners. As suggested from these available data and depending on further validations, new treatment strategies could be developed in order to broaden the use for PARP inhibitors in cancer patients

Therapeutic Perspectives for cN-II in Cancer.

International audienceThe cytoplasmic 5'-nucleotidase cN-II is involved in the regulation of endogenous pools of nucleosides and nucleotides together with nucleoside kinases and other intracellular enzymes. A series of results from studies on preclinical models and clinical samples constitutes the basis of the hypothesis in which cN-II is a therapeutic target in cancer. Indeed, the inhibition of its enzymatic activity seems interesting both to induce cell death directly and to increase the anticancer activity of cytotoxic agents used in cancer treatment. Here we will review the current knowledge of the enzymatic function of cN-II together with available structural data and the studies on cN-II in cancer cells and in samples from cancer patients. Recent and ongoing research on cN-II inhibitors is expected to confirm the druggability and the relevance of cN-II as a cancer drug target. Preliminary in vitro data and cancer cell models using cN-II inhibitors have already suggested the pivotal role of this enzyme as therapeutic target allowing the improvement of anticancer treatments

Réparation des cassures double brin d'ADN par recombinaison non homologue (rôle de la phosphorylation de Ku86 par la DNA-PK)

TOULOUSE3-BU Santé-Centrale (315552105) / SudocTOULOUSE3-BU Santé-Allées (315552109) / SudocSudocFranceF

Effet des adipocytes sur la progression tumorale de cellules cancéreuses d'origine hématopoïétique et rôle de l'adénosine

Il est aujourd'hui établi que l'obésité est un facteur de mauvais pronostic dans le cancer. L'interaction entre les adipocytes du microenvironnement tumoral et les cellules cancéreuses joue un rôle dans la progression tumorale, aussi bien dans la croissance et la migration tumorale que dans la résistance aux traitements. La compréhension des relations entre stroma et parenchyme tumoraux ainsi que la définition de la composition biochimique du microenvironnement tumoral sont des étapes nécessaires à l'élaboration de stratégies diagnostiques et thérapeutiques innovantes. La problématique de ce travail a été de savoir si l'adénosine produite par les adipocytes via l'ecto-5'-nucléotidase (CD73) pouvait faire partie intégrante du mécanisme de progression tumorale. Nous nous sommes intéressés aux cellules cancéreuses d'origine hématopoïétique puisque les adipocytes sont le constituant majoritaire du microenvironnement médullaire. Nous avons montré que les adipocytes favorisent la prolifération et la survie de cellules leucémiques et lymphomateuses in vitro. La caractérisation des expressions géniques et protéiques des récepteurs de l'adénosine par les cellules cancéreuses nous a permis d'attester leur capacité de réponse à l'adénosine. Nous avons alors objectivé une action cytostatique et cytotoxique de l'adénosine à des concentrations supraphysiologiques sur ces cellules cancéreuses. Ces résultats ont été obtenus dans des conditions expérimentales différentes ne permettant pas de répondre de manière satisfaisante à la question du rôle de l'adénosine dans l'effet des adipocytes. Des expériences de modulation de l'activité de l'enzyme CD73 fonctionnelle des adipocytes et de mesure de l'activation des récepteurs de l'adénosine ont suggéré que l'adénosine n'intervenait pas dans l'interaction entre les deux types cellulaires. La réponse définitive à cette problématique pourra sûrement être obtenue à l'aide d'un plasmide shRNACD73 obtenu au laboratoireLYON1-BU Santé (693882101) / SudocSudocFranceF

Un tour d’horizon du développement de biomarqueurs pour les anticorps PD-1 et PD-L1 en cancérologie

The immune checkpoints inhibitors targeting PD-1 or PD-L1 represent a new paradigm in the cancer treatment strategy. However, some populations of patients do not benefit from these agents. The identification of predictive biomarkers appears as an essential step for the treatment pathway, to guarantee the access to an evidence-based medicine accounting for the potential toxicity profile, the cost for the healthcare system and the clinical benefit eventually provided by these new drugs. In this review, we propose, based on scientific literature and industrial communications, an overview of the current landscape of predictive biomarkers related to PD-1 or PD-L1 inhibitors efficacy, validated or under development, their evidence level, and limits accounting for identified or potential confounding factors. Our paper shows that, despite the important amount of work performed in this field, there is not yet a validated and efficient solution for the prediction of the activity and/or the toxicity of anti-PD-1 and anti-PD-L1 antibodies

Étude de la résistance aux analogues de nucléosides cytotoxiques

Les analogues de nucléosides sont largement utilisés dans le traitement d'hémopathies malignes et certaines tumeurs solides. Des résistances primaires et acquises sont responsables d'une diminution d'activité cytotoxique de ces molécules. Notre équipe travaille depuis plusieurs années sur l'identification et l'étude des mécanismes responsables de ces résistances ainsi que sur le développement d'approches permettant de regagner une activité cytotoxique sur des cellules résistantes. Au cours de ce travail de thèse nous avons : (1) identifié un substrat de la 5'-nucléotidase cN-II parmi les analogues de nucléosides utilisés en clinique, et étudié la réaction enzymatique correspondante ; (2) développé et caractérisé plusieurs modèles cellulaires humains et murins résistants à la gemcitabine ; (3) étudié l'activité cytotoxique d'analogues de pronucléotides sur des modèles résistants par déficit en désoxycytidine kinase. Les résultats obtenus ont permis d'améliorer les connaissances de la résistance aux analogues de nucléosides cytotoxiques, et constituent la première étape de l'identification et le développement de molécules susceptibles de permettre le traitement de cellules résistantes aux analogues de nucléosidesLYON1-BU.Sciences (692662101) / SudocSudocFranceF