Inserm Institut national de la santé et de la recherche médicale Régulation de l’expression des enzymes du métabolisme des médicaments par les xénorécepteurs CAR et PXR et conséquences physiopathologiques. Jean Marc Pascussi Institut de Génomique Fonctionnelle Inserm U661, Montpellier [email protected] 1 Xenobiotics Xenobiotics: foreign chemicals Inhaled Pollutants Industrial chemicals Ingested absorbed Pesticides Toxins produced by molds, plants and animals Drugs Les xénorécepteurs, sentinelles moléculaires de l’immunité métabolique PXR Phase I métabolisme Phase II élimination AhR Phase III transporteurs CAR 6 Different strategies for activating transcription factors 8 9 Nuclear Hormone Receptors superfamily The Nuclear Receptor Gene superfamily 11 Nuclear receptors General scheme for activation of gene transcription by NRs The paradigm … Robyr, Wolffe, Wahli Mol. Endocrinol 2000 Les xénorécepteurs CAR et PXR Expression Organes de la détoxication: foie (hépatocytes) et tracus digestif (entérocytes) CAR PXR CAR and PXR coordinate the “metabolic immunity” in response to xenochemicals or endogenous activators Nuclear Receptor PXR (NR1I2) … the master xenosensor Nuclear receptor CAR (NR1I3) 17 Ligand CAR ou PXR Acide rétinoïque 9-cis RXR Gène cible Schéma de l’hétérodimère CAR-RXR ou PXR-RXR en présence de leurs ligands et différentes organisations des HREs. L’annotation x correspond au nombre de nucléotides entre les deux motifs (d’après Handschin C & Meyer UA. Pharmacol Rev, 2003; 55, 649-73) Les xénorécepteurs CAR et PXR partagent les mêmes NRE 2B6 3A4 2B6 3A4 CAR and PXR regulate the bodies 'garbage-disposal system.' Phase 0 Phase I Phase III Transport Uptake CYP2A,2B,3A Carboxylesterases GST SULT2 UGT1A PAPSSP2 MDR1A,1B MRP3… Solubilisation Phase II OATP2 excretion Les xénorécepteurs CAR et PXR Ligands/activateurs • PXR (Polygamic Xenobiotic Receptor ?) • CAR Médicaments: Antibiotiques (rifampicine,etc.) Glucocorticoides, RU486, PCN Antifongiques (clotrimazole) Anticancéreux (paclitaxel, codermolide, ixabepilone, tamoxifene, cyclophosphamide) Inhibiteur de protéases virales (amprenavir, ritonavir) Inhibiteurs des pompes à protons (Benzimidazoles) Bloqueurs des canaux calciques (nifedipine) Médicaments: Clotrimazole (agoniste inverse) Phenobarbital, phénytoïne Alimentation/Phytothérapie: Mille pertuis gluglustérone b-carotène Ginko Biloba CITCO (agoniste) Phytothérapies: Artémise Pesticides Pesticides Contaminants environnementaux rifampicine 823 g.mol-1 phénobarbital CITCO 232.17 g.mol-1 437 g.mol-1 hPXR LBD crystal structure (2.6 A resolution) Bound ligand= SR12813 Large and flexible ligand-binding pocket Presence of 2 additional strands of b-sheet Hydrophobic ligand-binding pocket Can accommodates a single hydrophobic ligand in multiple conformation Absence of a highly constrained pocket allows for molecular flexibility and plasticity in ligand recognition Watkins et al. Science 2001; Chrencik et al. Mol Endocrinol 2005 Plasticity in the PXR Binding Pocket SR12813 hyperforin 1280 Å Receptor ER VDR PXR CAR 1544 Å Volume of ligand pocket (A3) 476 871 1250-1550 1120 hCAR/RXR LBD heterodimer crystal structure (2.6 A resolution) RXR CAR Bound ligand: CITCO SRC-1 peptide Vol= 570-676 A Xu et al. Mol Cell 2004 CAR activators provoke CAR nuclear translocation CAR-GFP in human hepatocytes In vivo in mice NT PB CITCO Mécanismes d’activation des xénorécepteurs CAR et PXR Citco AMPc/AMP AICAR OA CAR PB ? AMPK LKB1 Thr-38 PP2A CCRP PKC CAR Thr-38-P cofactors RXR HSP CAR PXR PXR RIF cytoplasme 26 noyau Complementary roles of CAR and PXR toward xenobiotics recognition ? CAR PXR, CAR 28 29 Species differences in CAR and PXR activation Variation in LBD consistent with in vivo species differences in response to inducers (CYP3A1 DR3)2-tk-CAT CAR and PXR humanized mice Wolf et al. J Clin Invest. 2008 Ying and Yang of CAR and PXR RXR CAR/PXR Xenosensors that protect the body from a multitude of foreign chemicals (xenobiotics) and endogenous toxic compounds xenobiotic Drug-drug and food-drug interactions Interindividual variability in drug response Endocrine disruption VitD3, T3 and lipids metabolism Major Cytochrome P450s involved in the metabolism of clinically used drugs ... Metabolic Contribution hepatic only CYP 2C9 10% CYP 1A2 other 2% 3% CYP 3A4 CYP 2D6 CYP 2C9 CYP 1A2 other CYP 3A4 55% CYP 2D6 30% also small intestine ... are PXR and CAR target genes CAR and PXR are activated by widely used drugs and top selling phytochemicals Nuclear receptor Activating drugs PXR amprenavir, avasimibe, bosentan, carbamazepine, ciglitazone, clotrimazole, cortisone, corticosterone, cyclophosphamide, dexamethasone, efavirenz, exemestane, hydrocortisone, hyperforin, lovastatin, mifepristone, nelfinavir, nifedipine, omeprazole, paclitaxel, phenobarbital, phenytoin, rifabutin, rifampicin, ritonavir, simvastatin, spironolactone, tamoxifen, 4hydroxytamoxifen, troglitazone, troleandomycin, St John’s wort, Kava, Sophora flavescens CAR CITCO, phenobarbital, phenytoin Garlic, Ginkgo CAR and PXR xenosensors are involved in drug-drug interactions Drug A XeR RXR CYP enzymes CYPs Drug B OH Inactivation activation Xenosensors and drug-drug interactions «Fatal paracetamol poisoning in an epileptic (phenytoin)» Minton et al. 1988 Hum Toxicol «Loss of analgesic effect of morphine due to coadministration of rifampin » Fromm et al. 1997 Pain Xenosensors and diet-drug interactions «Profound drop of cyclosporin A whole blood through levels caused by St. John’s wort (Hypericum perforatum)» Breidenbach et al. 2000 Transplantation Role of CAR and PXR in drugs-induced osteomalacia and osteopenia ? «Rifampicin induced osteomalacia» Shah et al. 1981 Tubercle «Calcium metabolism during rifampicin and isoniazid therapy for tuberculosis» Brodie et al. 1982 J R Soc Med. «Osteomalacia associated with carbamazepine/valproate» Karaaslan et al. 2000 Ann Pharmacother «Antiretroviral therapy and the prevalence of osteopenia and osteoporosis : a meta-analytic review» Brown et al. 2006 AIDS. Déficits en vitamine D et ostéomalacies consécutives à la prise prolongée de médicaments… sur la piste de CAR et PXR 7 déhydroxycholestérol RXR PXR RXR VDR NRE CYP27A1 RXR CAR CYP3A4 (DR3, ER6) CYP2B6 (DR4) CYP2C9 (DR4) SULT2A1 (IR0) Thummel et al. Mol Pharm 2001 Drocourt et al. J Biol Chem 2002 Echchgadda et al. Mol Pharmacol 2004 25(OH)D3 CYP27B1 1,25(OH)D3 CYP24A1 1,24,25(OH)D3 (inactive) RXR VDR Les activateurs de CAR et PXR augmentent l’expression et l'activité de la CYP24 dans l’hépatocyte humain Pascussi , 2005, JCI Lambert 2008 TAP CYP24 : nouveau gène cible de CAR et PXR CAR et PXR transactivent les VDREs du gène CYP24 Konno 2008 Mol. Pharmacology HepG2 HuH7 HuH7 The VDR-PXR cross-talk CYP3A4 CYP24 XeR CYP3A4 CYP24 CYP3A4 CYP24 Drocourt 2002 JBC, Xu 2006 Mol P, Pascussi 2005 JCI, Moreau 2007 BBRC, K onno 2008 MP, Lambert 2008 TAP CAR, PXR et la stéatose hépatique non alcoolique induite par certains médicaments. - Rifampicine traitement contre la tuberculose Stéatose hépatique Morere 1975 Sem Hop - Nifedipine traitement contre l’hypertension Stéatose hépatique Babary 1989 J Hep - Carbamazepine traitement antiépileptique Stéatose hépatique Oscarson 2006 CPT - Phenobarbital traitement antiépileptique Stéatose hépatique Calandre 1991 ANS Nakamura 2007 JBC Etudes comparatives de transcriptomes d’hépatocytes humains ... sur la piste d’un acteur peu connu de la lipogénese : Spot14 Culture primaire d’hépatocytes humains Extraction ARN Traitements (8 & 40h): DMSO 0.1% Phénobarbital (0.5mM) Rifampicine (10M) CITCO (100nM) Protocole double couleur - ARN de cultures traitées - ARN de référence Spot14, THRSP (Chr. 11q13.5/14.1) 143AA, 17kDa, Pi=4.75 Les activateurs de PXR augmentent l’expression de Spot14 dans l’hépatocyte humain. FT285 mRNA fold induction 30 25 Actin CYP3A4 CYP2B6 S14 CTRL Rif 0,3 (PXR) 20 Rif 3 (PXR) 15 Rif 30 (PXR) SR12813 (PXR) 10 5 PAX (LXR) T09 (PXR + LXR) T3 (T3R) Spot14 est un gène cible de PXR SFN L’élément TRE de promoteur du gène Spot14 est nécessaire à l’action de CAR et PXR Les activateurs de PXR augmentent l’expression de la FASN dans l’hépatocyte humain. L’expression de Spot14 est nécessaire à l’induction de la FASN par PXR L’activation de PXR provoque une accumulation d’acides gras dans l’hépatocyte humain. Analyses lipidomiques: Quantification des acides gras dans les hépatocytes humains chromatographie liquide à ultra haute performance (UPLC) et spectrométrie de masse (Q-TRAP) nmoles/g protéines nmoles/g protéines +/- RIF (72h) L’activation de PXR provoque une augmentation de la lipogenèse de novo Michel Beylot INSERM ERI22 Mesure de la lipogenèse de novo : Quantification de l’incorporation de deutérium dans C16:0 par chromatographie en phase gazeuse et spectrométrie de masse 3% D2O (6h) Palmitate IE MPE +/- RIF (72h) Ectopic overexpression of S14 increases lipogenesis in HepaRG cells 27mM Glucose FG12 hS14 Palmitate IE MPE De novo lipogenesis FG12 hS14 6mM Glucose FA quantifications FG12 En conclusion: L’activation des xénorécepteurs induit une forte perturbation du métabolisme lipidique provoquant une accumulation des acides gras et des triglycérides hépatiques. Role of CAR and PXR in interindividual variability in response to cancer therapy ? XS Narrow therapeutic index Toxicity Efficacy CAR and PXR regulate genes involved in biotransformation and clearance of widely used anticancer drugs CYP2A6 cyclophosphamide, ifosmamide, flutamine, tegafur CYP2B6 altretamine, cyclophosphamide, ifosmamide, tomoxifen UGT doxorubicine, epirubicin, etoposide, cyclophosphamide, docetaxel, ifosmamide, paclitaxel, tegafur, tretinoin SULT tamoxifen CYP2C8 CYP3A4/5 irinotecan, topotecan, tamoxifen GST busulfan, chlorambucil, cyclophosphamide, doxorubicin, Bexarotene, busulfan, cisplatin, ifosphamide, melphalan, nitrosurea cyclophosphamide, cytarabine, dexamethasone, docetaxel, doxorubicin, erlotinib, etoposide, exemestane, flutamide,fulvestrant, gefitinib, ifosfamide, imatinib, irinotecan, letrozole, medroxyprogresterone mitoxantrone, paclitaxel, targretin, teniposide, acetate, tamoxifen, topotecan, toremifene, tretinoin, vinblastine, vincristine, vindesine, vinorelbine MRP1 Arsenic trioxide, chlorambucil, daunorubicin, doxorubicin, epirubicin, etoposide, melphalan, methotrexate, mitoxantrone, paclitaxel, vinblastine, vincristine MRP2 Cisplatin, irinotecan doxorubicin, etoposide, methotrexate, SN-38, vinblastine, vincristine MRP3 Carboplatin, cisplatin, doxorubicin, epirubicin, etoposide, methotrexate, teniposide, vinblastine, vincristine BCRP Imatinib, methotrexate, mitoxantrone, SN-38, topotecan Role of CAR and PXR in pharmacokinetic drug-drug interaction in oncology, some examples Rifampicin Phenytoin phenobarbital Decrease cyclophosphamide and increase 4-hydroxyclyclophosphamide plasma concentrations Severe toxicity Therapeutic escape and risk of relapse. Rifampicin Phenobarbital Phenytoin St John’s wort rifampicin Decrease CPT11 & SN38 and increase SN38-G plasma concentrations Increases of erlotinib clearance and reduces the AUC by 66¨% Role of CAR and PXR in cyclophosphamide activation phenytoin phenytoin Chang, Yu, Maurel, Waxman. Cancer Res., 1997 De Jonge, Cancer Chemother Pharmacol. 2005 Role of CAR and PXR on peripheral metabolism of irinotecan (Campto ®) CAR and PXR are expressed in several neoplastic human tissues Neuroblastoma (PXR) Misawa , Cancer Res, 2005 Endomedrial cancer cells (PXR) Masuyama, Mol. Pharm., 2007 Hepatocarcinoma (CAR, PXR) Huang , Mol Endocrinol, 2005 Pascussi Hepatology 2007 Intestinal & colon cancer cells (CAR, PXR) Jiang, J Gastrointest Surg, 2009 Raynal, Mol.Cancer, 2010 Ouyang, Br J Cancer, 2010 Breast tissues (PXR) Dotzlaw, 1999 Miki, Cancer Res, 2006 Lung cancer cells (PXR) Miki, Mol Cell Endocrinol, 2005 Ovarian cancer tissues (PXR) Gupta, Human Cnacer Biology , 2008 Osteosarcoma (PXR) Mensah-Osman, Cancer, 2007 Prostate cancer cells (PXR) Chen, Cancer res., 2008 Expression of PXR in Human Breast Carcinoma LCM/RT-PCR (C) carcinoma cells (S) stromal cells PXR OATP-A Miki et al, Cancer Res 2006; 66: (1). January 1, 2006 PXR expression in normal and cancerous human prostate tissues Chen Y et al., Cancer Res 2007;67:10361-10367 Increased chemoresistance in PC-3 cells by PXR agonist, SR12813. CTRL 0.2 μM SR12813 1 μM SR12813 Chen Y et al., Cancer Res 2007;67:10361-10367 Increased chemosensitivity of PC-3 cells with PXR expression knocked down Chen Y et al., Cancer Res 2007;67:10361-10367 Increased chemosensitivity of endothelial HEC-1cells with PXR expression knocked down Masuyama, Mol. Pharm., 2007 Increased chemoresistance in ovarian carcinoma cells by PXR agonist SKOV-3 D. Gupta et al. Human cancer Biology, 2008 Role of CAR and PXR in intra-tumoral metabolism of irinotecan ? Systemic drug clearance Cancer cells ? PXR expression in normal and cancerous human colon tissues Raynal C. et al. Mol. Cancer Res 2010 PXR expression level restricts SN38 chemosensibility in colon cancer cells LS174T SW620 PXR expression level restricts SN38 chemosensibility in colon cancer cells PXR expression level enhances SN38 glucuronidation in colon cancer cells Intracellular metabolites profiles (HPLC PXR increases UGT1As-mediated SN38 inactivation in colon cancer cells SN38 (cytotoxic metabolite) PXR UGT1As SN38-G (inactive metabolite) SN38 SN38-G CAR/PXR UGT1A1 SN38 (cytotoxic metabolite) SN38-G (inactive metabolite) Role of CAR and PXR in intra-tumoral metabolism of cyclophosphamide ? Pro-drug activation Cancer cells ? PXR regulates ALDH1A1 gene expression and Aldefluor® activity CAR and PXR may increase cancer cell resistance to 4-OH-CPA while promoting severe toxicity Hepatic cells Cancer cells a Aldefluor®-positive cells are associated with cancer initiation properties, chemotherapy-resistance and poor clinical outcome Aldehyde Dehydrogenase 1 is a marker for normal and malignant human colonic stem cells and have tumor initiating properties CD44 ALDH1 Nuclei Aldefluor®-positive Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy PXR NANOG BMI1 CD26 LGR5 OCT4 CYP3A4 ABCG2 ALDH1A1 PXR Aldefluor®-positive cells display higher expression of PXR and PXR target genes Aldefluor®-positive cells display enhanced clonogenic, sphere forming activity and magnétorésistance Nb sphere / 100 cells * ALDHlow ALDHbr Soft-agar (1000 cells) – 3 weeks PXR knock-down decreases chemoresistance of Aldefluor®-positive cells siRNA (sibGAL / siPXR) 100nM Cell viability (ATP content) PXR knock-down decreases xenogeneic tumor recurrence Tumor formation Folfiri: 50mg/kg 5-FU 30mg/kg irinotecan Treatment response Relapse ONCO TALK - 29/01/2013 PXR knock-down decreases Aldefluor®-positive cells enrichment and tumor initiating activity after cytotoxics treatment 15.000 cells 3D FOLFIRI Tumor cells dissociation reinjection 1500 cells PXR inhibition as a new strategy for Chemoresistant and Tumor Initiating Cells (CTIC) re-sensitization to conventional therapies ? Conventional therapy PXR inhibition & conventional therapy Les xénorécepteurs CAR et PXR Gènes Cibles Enzymes et transpoteurs majeurs de la fonction de détoxication entérohépatique OATP2 CYP3A4,5,7 CYP2B6 CYP2C8,9 CYP1A2 UGT1A1,6,9 GSTs SULT2A1 MDR1 MRP2-4 Interactions croisées avec d’autres voies Perturbations endocriniennes - Vitamine D Perturbations métaboliques - Lipides cellulaires CAR/PXR Adduit Xénobiotique Transporteurs CYP450 Molécule thérapeutique Interactions médicamenteuses Transférases Métabolites - Hépatite fulminante - HCC inactif actif 89 90 Conclusion Cells and organisms are able to increase and adapt their capacity of detoxication in response to some xenobiotics and drugs Nuclear receptors PXR and CAR play a major part in this process by controlling a network of signaling pathways that regulate the expression of specific batteries of genes involved in the detoxication machinery Because 1) many endocrine hormones are metabolised by CAR and PXR target genes, and 2) they interfere with other signalling pathways, chronic activation of these NRs (drugs, industrial or natural contaminants) could alter endocrine physiology and disease promotion. According to their role as masters xenobiotic responsive receptors linking DME genes expression to environment stimuli, CAR and PXR might contribute to the well-known intraand inter-subject variability in anticancer drugs response. Environmental and genetic factors affecting CAR or PXR (expression or activation levels) may affect the cytotoxic threshold of tumor cells to chemotherapy which can consequently mask or attenuate pharmacogenetic associations.