Etienne Blanc

Low expression of Wnt-5a gene is associated with high-risk neuroblastoma

Disseminated forms of neuroblastoma (NB), a tumor derived from neuroectodermal tissue, pose a major therapeutic challenge for pediatric oncology. By performing a comparative cDNA array analysis of metastatic neuroblasts versus primary xenograft from the human IGR-N-91 NB model, we were able to identify a set of downregulated developmental genes in metastatic neuroblasts. One of these genes was Wnt-5a, a member of the Wnt signaling pathway, known to be involved in the development of neural crest cells. Since we also found a significant decrease in Wnt-5a mRNA in unfavorable versus favorable categories in 37 primary NB tumors (P<0.007), we wondered whether retinoic acid (RA), which has a role in neural crest induction and differentiation, might reverse the aberrant negative regulation of Wnt-5a in metastatic malignant neuroblasts. Following treatment with 10 μM RA for 6 days, the MYCN-amplified IGR-N-91 cell lines underwent neuronal differentiation as assessed by reduced MYCN gene expression and neuritic extension. In these conditions, data showed an upregulation of Wnt-5a and PKC-θ isoform expressions. Our study highlights, for the first time, the involvement of Wnt-5a, which has a role in embryonic and morphogenetic processes, in the response of malignant neuroblasts to RA. In conclusion, we demonstrated that RA, which is used in the treatment of high-risk NB patients with recurrent/residual disease in the bone marrow, is able to upregulate Wnt-5a gene expression.

Hypoxia Down-regulates CCAAT/Enhancer Binding Protein-α Expression in Breast Cancer Cells

The transcription factor CCAAT/enhancer binding protein-alpha (C/EBP alpha) is involved in the control of cell differentiation and proliferation, and has been suggested to act as a tumor suppressor in several cancers. By using microarray analysis, we have previously shown that hypoxia and estrogen down-regulate C/EBP alpha mRNA in T-47D breast cancer cells. Here, we have examined the mechanism by which the down-regulation by hypoxia takes place. Using the specific RNA polymerase II inhibitor 5,6-dichlorobenzimidazole-1-beta-D-ribofuranoside, the mRNA stability was analyzed under normoxia or hypoxia by quantitative reverse transcription-PCR. Hypoxia reduced the half-life of C/EBP alpha mRNA by approximately 30%. C/EBP alpha gene promoter studies indicated that hypoxia also repressed the transcription of the gene and identified a hypoxia-responsive element (-522; -527 bp), which binds to hypoxia-inducible factor (HIF)-1 alpha, as essential for down-regulation of C/EBP alpha transcription in hypoxia. Immunocytochemical analysis showed that C/EBP alpha was localized in the nucleus at 21% O(2), but was mostly cytoplasmic under 1% O(2). Knockdown of HIF-1 alpha by RNAi restored C/EBP alpha to normal levels under hypoxic conditions. Immunohistochemical studies of 10 tumor samples did not show any colocalization of C/EBP alpha and glucose transporter 1 (used as a marker for hypoxia). Taken together, these results show that hypoxia down-regulates C/EBP alpha expression in breast cancer cells by several mechanisms, including transcriptional and posttranscriptional effects. The down-regulation of C/EBP alpha in hypoxia is mediated by HIF-1.

ATF4 and the integrated stress response are induced by ethanol and cytochrome P450 2E1 in human hepatocytes

BACKGROUND & AIMS Molecular mechanisms underlying alcoholic liver disease (ALD) are still not fully understood. Activating transcription factor-4 (ATF4) is the master coordinator of the integrated stress response (ISR), an adaptive pathway triggered by multiple stressors. which can promote cell death and induce metabolic dysregulation if the stress is intense or prolonged. The aim of this study was to assess the effect of alcohol on the ISR signaling pathway in human liver cells and to define the role of cytochrome P450 2E1 (CYP2E1) in this response. METHODS Primary cultured human hepatocytes and human HepG2 cells over-expressing CYP2E1 by adenoviral infection were exposed to ethanol (25-100mM) for 8-48h. RESULTS Ethanol treatment of both liver cells up-regulated ATF4 as well as the pro-survival and the pro-apoptotic transcriptional program of the ISR. Indeed, in CYP2E1-expressing HepG2 cells exposed to ethanol, the expression of ISR target genes (HMOX-1, GCLC, AsnS, IGFBP-1, GADD34,CHOP, ATF3, CHAC1) was induced. Up-regulation of ATF4 and the ISR transcriptional program was decreased by addition of the anti-oxidant glutathione. Several mechanisms mediated ATF4 protein induction, including, at early times, the phosphorylation of eIF2α which controls ATF4 translation, and, at later times, increased mRNA level and increased stability of the protein. A decrease in cell survival was also observed. CONCLUSIONS This study demonstrates that both CYP2E1 and ethanol induce ATF4 and the integrated stress response, a pathway which coordinates signals from multiple stresses, as well as established risk factors for ALD, and can display detrimental cellular effects upon prolonged activation.

Differential response of p53 target genes to p73 overexpression in SH-SY5Y neuroblastoma cell line

p73, the first p53 gene homologue, encodes an array of p73 proteins including p73α full-length (TAp73α) and amino-truncated isoforms (ΔNp73α), two proteins with opposite biological functions. TAp73α can induce tumor suppressive properties, while ΔNp73α antagonizes p53 as well as TAp73 in a dominant-negative manner. In human malignant neuroblasts, p53 protein is wild-type but known to be excluded from the nucleus, therefore disabling its function as a tumor suppressor. The present study investigates whether there is a functional link between p73 isoforms and p53 in neuroblastoma. Experiments were performed on two neuroblastoma cell lines differing in their p53 status, e.g. wild-type p53 SH-5Y5Y cells and mutated p53 IGR-N-91 cells. Data indicate that (i) both TA- and ΔN-p73α enhance p53 protein level in SH-SY5Y cells, whereas level remains unchanged in IGR-N-91 cells; (ii) only in SH-SY5Y cells does forced TAp73α overexpression markedly induce nuclear accumulation of p53 protein; (iii) p21 protein expression is increased in both cell lines infected with TAp73, suggesting that, in IGR-N-91 cells, p21 is induced by p73 through a p53-independent pathway; (iv) in the SHSY5Y cell line, Btg2 expression is strongly enhanced in cells overexpressing TA, and to a lesser extent in cells overexpressing ΔN. Taken together our results suggest that TAp73 may restore p53 function in NB with wild-type nonfunctional p53, but not in NB with mutated p53.

Down-regulation of the expression of alcohol dehydrogenase 4 and CYP2E1 by the combination of α-endosulfan and dioxin in HepaRG human cells

Pesticides and other persistent organic pollutants are considered as risk factors for liver diseases. We treated the human hepatic cell line HepaRG with both 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) and the organochlorine pesticide, α-endosulfan, to evaluate their combined impact on the expression of hepatic genes involved in alcohol metabolism. We show that the combination of the two pollutants (25nM TCDD and 10μM α-endosulfan) led to marked decreases in the amounts of both the mRNA (up to 90%) and protein (up to 60%) of ADH4 and CYP2E1. Similar results were obtained following 24h or 8days of treatment with lower concentrations of these pollutants. Experiments with siRNA and AHR agonists and antagonist demonstrated that the genomic AHR/ARNT pathway is necessary for the dioxin effect. The PXR, CAR and estrogen receptor alpha transcription factors were not modulators of the effects of α-endosulfan, as assessed by siRNA transfection. In another human hepatic cell line, HepG2, TCDD decreased the expression of ADH4 and CYP2E1 mRNAs whereas α-endosulfan had no effect on these genes. Our results demonstrate that exposure to a mixture of pollutants may deregulate hepatic metabolism.

Impact of Dyrk1A level on alcohol metabolism

Alcoholic liver diseases arise from complex phenotypes involving many genetic factors. It is quite common to find hyperhomocysteinemia in chronic alcoholic liver diseases, mainly due to deregulation of hepatic homocysteine metabolism. Dyrk1A, involved in homocysteine metabolism at different crossroads, is decreased in liver of hyperhomocysteinemic mice. Here, we hypothesized that Dyrk1A contributes to alcohol-induced hepatic impairment in mice. Control, hyperhomocysteinemic and mice overexpressing Dyrk1A were fed using a Lieber-DeCarli liquid diet with or without ethanol (5% v/v ethanol) for one month, and liver histological examination and liver biochemical function tests were performed. Plasma alanine aminotransferase and homocysteine levels were significantly decreased in mice overexpressing Dyrk1A compared to control mice with or without alcohol administration. On the contrary, the mean plasma alanine aminotransferase and homocysteine levels were significantly higher in hyperhomocysteinemic mice than that of control mice after alcohol administration. Paraoxonase 1 and CYP2E1, two phase I xenobiotic metabolizing enzymes, were found increased in the three groups of mice after alcohol administration. However, NQO1, a phase II enzyme, was only found increased in hyperhomocysteinemic mice after alcohol exposure, suggesting a greater effect of alcohol in liver of hyperhomocysteinemic mice. We observed positive correlations between hepatic alcohol dehydrogenase activity, Dyrk1A and ADH4 protein levels. Importantly, a deleterious effect of alcohol consumption on hepatic Dyrk1A protein level was found. Our study reveals on the one hand a role of Dyrk1A in ethanol metabolism and on the other hand a deleterious effect of alcohol administration on hepatic Dyrk1A level.

P020: Interaction entre la réponse aux polluants environnementaux et les enzymes du métabolisme de l’alcool : rôle dans les pathologies hépatiques

Introduction et but de l’etude Les populations des pays industrialises et en voie de developpement sont communement, et de plus en plus, exposees a divers polluants environnementaux. Parmi eux, les « polluants organiques persistants » (POP) sont caracterises par une longue demi-vie due a un metabolisme tres lent et a leur hydrophobicite, qui contribuent ainsi a leur stockage dans les graisses et a leur accumulation le long de la chaine alimentaire. Des etudes epidemiologiques recentes montrent que l’exposition chronique a de faibles doses de POP (en particulier les pesticides organochlores et les polychlorobiphenyles (PCBs) mais aussi les dioxines) pourrait jouer un role important, mais encore mal caracterise dans l’initiation et/ou la progression tumorale. Tres recemment, notre laboratoire a montre pour la premiere fois que la TCDD (2,3,7,8-TetraChloroDibenzo- para-Dioxine, dioxine la plus toxique) pouvait induire une fibrose hepatique chez la souris (Pierre et al, 2014), et nous cherchons a comprendre les mecanismes qui menent au developpement de cette pathologie voire a sa progression vers le cancer du foie. Materiel et methodes Le modele que nous utilisons est la lignee cellulaire HepaRG, issue d’un hepatocarcinome humain, qui presente la caracteristique de se differencier en presence de DMSO 1,5 % pendant 15 jours, en deux types cellulaires, biliaire et hepatocytaire. Il represente a l’heure actuelle, l’un des meilleurs modeles hepatocellulaires humains. Apres differenciation, les cellules sont traitees avec de la TCDD, un polluant de la famille des composes poly-aromatiques halogenes, qui utilise la voie de signalisation du AhR (recepteur aux hydrocarbures aromatiques), ainsi qu’avec d’autres ligands de ce meme recepteur (3-methylcolanthrene, PCB153). Resultats et Analyse statistique Une etude transcriptomique (puces a ADN) realisee au laboratoire sur des cellules HepaRG differenciees, a montre que la TCDD diminue de maniere drastique l’expression de certains genes du metabolisme intermediaire, et en particulier les genes codant les enzymes du metabolisme de l’alcool [diminution d’environ 80-90 % de l’expression des ARNm des CYP2E1, ADH1A-B-C (Alcool DesHydrogenase, classe I), ADH4 (classe II) et ADH6 (classe V)]. Ces enzymes sont aussi impliquees dans le metabolisme des retinoides (vitamine A), molecules qui jouent un role crucial dans le maintien d’une activite cellulaire normale. L’etude des mecanismes moleculaires de cette regulation montre qu’apres un traitement de 25nM de TCDD, le niveau d’expression des ARNm de ces enzymes diminue significativement des 8h, et apres 72h au niveau proteique. Lors d’un traitement de 24 h, cette regulation apparait neanmoins pour des concentrations de 0,1nM. La mesure de la demi-vie de l’ARNm de ces enzymes indique que leur regulation se fait au niveau transcriptionnel. Enfin, l’utilisation d’inhibiteurs pharmacologiques, de siARN et de differents ligands du AhR, montre l’implication de ce recepteur et de son partenaire nucleaire ARNT dans cette regulation. Conclusion Nos resultats suggerent que la TCDD, ainsi que d’autres POP, ligands du AhR, inhibent l’expression des enzymes du metabolisme de l’alcool via l’activation du AhR et une regulation transcriptionnelle. Nos perspectives sont 1) d’approfondir les mecanismes moleculaires de cette regulation et en particulier de savoir s’il s’agit d’une regulation directe ; 2) d’analyser les consequences de ces regulations sur le metabolisme des retinoides et le processus de cancerogenese ou de progression tumorale.