1 régulation de lexpression des enzymes du métabolisme des médicaments par les xénorécepteurs...

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Régulation de l’expression des enzymes du métabolisme des médicaments par les Régulation de l’expression des enzymes du métabolisme des médicaments par les xénorécepteurs CAR et PXR et conséquences physiopathologiques.xénorécepteurs CAR et PXR et conséquences physiopathologiques.

Jean Marc PascussiJean Marc PascussiInstitut de Génomique FonctionnelleInstitut de Génomique Fonctionnelle

Inserm U661, MontpellierInserm U661, MontpellierJean-marc.pascussi@inserm.frJean-marc.pascussi@inserm.fr

InsermInsermInstitut nationalInstitut nationalde la santé et de la recherche médicalede la santé et de la recherche médicale

Xenobiotics

Xenobiotics: foreign chemicals

Inhaled

Ingested

absorbed

Pollutants

Industrial chemicals

Pesticides

Toxins produced by molds, plants and animals

Drugs

Les xénorécepteurs, sentinelles moléculaires de l’immunité métabolique

PXRPXR

AhRAhR

CARCAR

Phase IPhase I

Phase IIPhase II

Phase IIIPhase IIItransporteurstransporteurs

métabolisme

élimination

6

Different strategies for activating transcription factors

8

9

Nuclear Hormone Receptors superfamily

11

The Nuclear Receptor Gene superfamily

Nuclear receptors

The paradigm …

General scheme for activation of gene transcription by NRs

Robyr, Wolffe, Wahli Mol. Endocrinol 2000

Les xénorécepteurs CAR et PXR

ExpressionExpression

Organes de la détoxication: foie (hépatocytes) et tracus digestif (entérocytes)

PXRCAR

CAR and PXR coordinate the “metabolic immunity” in response to xenochemicals or endogenous activators

Nuclear Receptor PXR (NR1I2) … the master xenosensor

17

Nuclear receptor CAR (NR1I3)

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)

CAR ou PXR RXR

Acide rétinoïque 9-cisLigand

Gène cible

Les xénorécepteurs CAR et PXR partagent les mêmes NRE

2B6 3A4 2B6 3A4

CAR and PXR regulate the bodies 'garbage-disposal system.'

excretion

Uptake

Solubilisation

Phase I CYP2A,2B,3ACarboxylesterases

Phase IIGST

SULT2UGT1A

PAPSSP2

Phase IIITransport

MDR1A,1BMRP3…

Phase 0 OATP2

Les xénorécepteurs CAR et PXR

CITCO437 g.mol-1

823 g.mol-1

Ligands/activateursLigands/activateurs

• PXR (PXR (Polygamic Xenobiotic Receptor ?)Polygamic Xenobiotic Receptor ?)

Médicaments:Antibiotiques (rifampicine,etc.)Glucocorticoides, RU486, PCNAntifongiques (clotrimazole)Anticancéreux (paclitaxel, codermolide, ixabepilone, tamoxifene, cyclophosphamide)Inhibiteur de protéases virales (amprenavir, ritonavir)Inhibiteurs des pompes à protons (Benzimidazoles)Bloqueurs des canaux calciques (nifedipine)

Alimentation/Phytothérapie: Mille pertuisgluglustéroneb-carotèneGinko Biloba

Pesticides

Contaminants environnementaux

• CARCAR

Médicaments:Clotrimazole (agoniste inverse)Phenobarbital, phénytoïne

Phytothérapies:Artémise

Pesticides

CITCO (agoniste)

phénobarbital

232.17 g.mol-1rifampicine

hPXR LBD crystal structure(2.6 A resolution)

Watkins et al. Science 2001; Chrencik et al. Mol Endocrinol 2005Watkins et al. Science 2001; Chrencik et al. Mol Endocrinol 2005

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

Bound ligand= SR12813

Plasticity in the PXR Binding Pocket

hyperforinSR12813

1280 Å 1544 Å

Receptor Volume of ligand pocket (A3)

ER 476VDR 871PXR 1250-1550CAR 1120

hCAR/RXR LBD heterodimer crystal structure(2.6 A resolution)

Bound ligand:CITCO

RXRRXR CARCAR

Xu et al. Mol Cell 2004Xu et al. Mol Cell 2004

SRC-1 peptideSRC-1 peptide

Vol= 570-676 A

CAR activators provoke CAR nuclear translocation

NT

PB

CITCO

CAR-GFP in human hepatocytesIn vivo in mice

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Mécanismes d’activation des xénorécepteurs CAR et PXR

CARCARPXRPXR

RXRRXR

cofactors

CARCAR

cytoplasmecytoplasme noyaunoyau

PBPBCCRPCCRP

OA AMPc/AMPAICAR

AMPKLKB1

HSPHSP

CARCAR

PXRPXRRIF

CitcoCitco

Thr-38-P

Thr-38

PP2APP2A

PKC

??

CAR

PXR, CAR

Complementary roles of CAR and PXR toward xenobiotics recognition ?

28

29

Species differences in CAR and PXR activation

(CYP3A1 DR3)2-tk-CAT

Variation in LBD consistent with in vivo species differences in response to inducers

CAR and PXR humanized mice

Wolf et al. J Clin Invest. 2008

xenobiotic

RXR CAR/PXR

Endocrine disruptionVitD3, T3 and lipids metabolism

Xenosensors that protect the body from a multitude of foreign chemicals (xenobiotics) and endogenous toxic compounds

Drug-drug and food-drug interactionsInterindividual variability in drug response

Ying and Yang of CAR and PXR

Metabolic Contribution

CYP 2D630%

CYP 1A22%CYP 2C9

10%

other3%

CYP 3A455%

CYP 3A4

CYP 2D6

CYP 2C9

CYP 1A2

other

hepatic only

also small intestine

Major Cytochrome P450s involved in the metabolism of

clinically used drugs ...

.... are PXR and CAR target genes

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, 4-hydroxytamoxifen,

troglitazone, troleandomycin, St John’s wort, Kava, Sophora flavescens

CAR CITCO, phenobarbital, phenytoinGarlic, Ginkgo

CAR and PXR are activated by widely used drugs and top selling phytochemicals

CAR and PXR xenosensors are involved in drug-drug interactions

CYPsCYP

enzymes

XeR RXRDrug A

OHDrug B Inactivation

activation

Xenosensors and drug-drug interactions

«Loss of analgesic effect of morphine due to coadministration of rifampin » Fromm et al. 1997 Pain

«Fatal paracetamol poisoning in an epileptic (phenytoin)» Minton et al. 1988 Hum Toxicol

«Profound drop of cyclosporin A whole blood through levels caused by St. John’s wort (Hypericum perforatum)» Breidenbach et al. 2000 Transplantation

Xenosensors and diet-drug interactions

«Osteomalacia associated with carbamazepine/valproate» Karaaslan et al. 2000 Ann Pharmacother

«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.

«Antiretroviral therapy and the prevalence of osteopenia and osteoporosis : a meta-analytic review»Brown et al. 2006 AIDS.

Role of CAR and PXR in drugs-induced osteomalacia and osteopenia ?

CYP3A4 (DR3, ER6)CYP2B6 (DR4)CYP2C9 (DR4)SULT2A1 (IR0)

PXRPXRRXRRXR VDRVDRRXRRXR RXRRXR CARCAR

NRENRE

Thummel et al. Mol Pharm 2001

Drocourt et al. J Biol Chem 2002

Echchgadda et al. Mol Pharmacol 2004

25(OH)D3

1,25(OH)D3

1,24,25(OH)D3(inactive)

7 déhydroxycholestérol

CYP27A1

CYP27B1

CYP24A1

VDRVDRRXRRXR

Déficits en vitamine D et ostéomalacies consécutives à la prise prolongée de médicaments… sur la piste de CAR et PXR

Les activateurs de CAR et PXR augmentent l’expression et l'activité de la CYP24 dans l’hépatocyte humain

Lambert 2008 TAP

Pascussi , 2005, JCI

CYP24 : nouveau gène cible de CAR et PXR

HepG2 HuH7

CAR et PXR transactivent les VDREs du gène CYP24

Konno 2008 Mol. Pharmacology

HuH7

The VDR-PXR cross-talk

CYP3A4CYP24

CYP3A4CYP24

XeR

CYP3A4CYP24

Drocourt 2002 JBC, Xu 2006 Mol P, Pascussi 2005 JCI, Moreau 2007 BBRC, Konno 2008 MP, Lambert 2008 TAP

CAR, PXR et la stéatose hépatique non alcoolique induite par certainsmédicaments.

Nakamura 2007 JBC

- Rifampicine traitement contre la tuberculose Stéatose hépatique Morere 1975 Sem Hop

- Carbamazepine traitement antiépileptique Stéatose hépatique Oscarson 2006 CPT

- Phenobarbital traitement antiépileptique Stéatose hépatique Calandre 1991 ANS

- Nifedipine traitement contre l’hypertensionStéatose hépatique Babary 1989 J Hep

Protocole double couleur - ARN de cultures traitées - ARN de référence

Extraction ARN

Culture primaire d’hépatocytes humains

Traitements (8 & 40h):DMSO 0.1%Phénobarbital (0.5mM)Rifampicine (10M)CITCO (100nM)

Etudes comparatives de transcriptomes d’hépatocytes humains... sur la piste d’un acteur peu connu de la lipogénese : Spot14

Spot14, THRSP(Chr. 11q13.5/14.1)

143AA, 17kDa, Pi=4.75

5

10

15

20

25

30 Actin CYP3A4 CYP2B6 S14

CTRL

Rif 0,3 (PXR)

Rif 3 (PXR)

Rif 30 (PXR)

SR12813 (PXR)

PAX (LXR)

T09 (PXR + LXR)

T3 (T3R)

FT

285

mR

NA

fol

d in

duct

ion

Les activateurs de PXR augmentent l’expression de Spot14 dans l’hépatocyte humain.

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 grasdans 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)

nm

oles

/g p

roté

ines

nm

oles

/g p

roté

ines

+/- RIF (72h)

Palm

itate

IE M

PE

+/- RIF (72h)

3% D2O (6h)

Mesure de la lipogenèse de novo :Quantification de l’incorporation de deutérium dans C16:0 par chromatographie en phase gazeuse et spectrométrie de masse

L’activation de PXR provoque une augmentation de la lipogenèse de novo

Michel BeylotINSERM ERI22

FG12 hS14

Ectopic overexpression of S14 increases lipogenesis in HepaRG cells

Palm

itate

IE M

PE

FG12 hS14 FG12

6mM Glucose

27m

M G

luco

se

FA quantifications

De novo lipogenesis

En conclusion:

L’activation des xénorécepteurs induit une forte perturbation du métabolisme lipidique provoquant une accumulation des acides gras et des triglycérides hépatiques.

XS

Role of CAR and PXR in interindividual variability in response to cancer therapy ?

Toxicity

Efficacy

Narrow therapeutic index

CAR and PXR regulate genes involved in biotransformation and clearance of widely used anticancer drugs

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

CYP2A6 cyclophosphamide, ifosmamide, flutamine, tegafur

CYP2B6 altretamine, cyclophosphamide, ifosmamide, tomoxifen

CYP2C8 cyclophosphamide, docetaxel, ifosmamide, paclitaxel, tegafur, tretinoin

CYP3A4/5 Bexarotene, busulfan, cisplatin, cyclophosphamide, cytarabine, dexamethasone, docetaxel, doxorubicin, erlotinib, etoposide, exemestane, flutamide,fulvestrant, gefitinib, ifosfamide, imatinib, irinotecan, letrozole, medroxyprogresterone acetate, mitoxantrone, paclitaxel, tamoxifen, targretin, teniposide, topotecan,toremifene, tretinoin, vinblastine, vincristine, vindesine, vinorelbine

UGT doxorubicine, epirubicin, etoposide, irinotecan, topotecan, tamoxifen

SULT tamoxifen

GST busulfan, chlorambucil, cyclophosphamide, doxorubicin, ifosphamide, melphalan, nitrosurea

Role of CAR and PXR in pharmacokinetic drug-drug interaction in oncology, some examples

Rifampicin Phenobarbital Phenytoin Decrease CPT11 & SN38 and increase SN38-G plasma concentrations St John’s wort

RifampicinPhenytoin Decrease cyclophosphamide and increase 4-hydroxyclyclophosphamide plasma concentrationsphenobarbital

Severe toxicity

Therapeutic escape and risk of relapse.

rifampicin Increases of erlotinib clearance and reduces the AUC by 66¨%

Role of CAR and PXR in cyclophosphamide activation

Chang, Yu, Maurel, Waxman. Cancer Res., 1997De Jonge, Cancer Chemother Pharmacol. 2005

phenytoin

phenytoin

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, 2009Raynal, Mol.Cancer, 2010Ouyang, Br J Cancer, 2010

Breast tissues (PXR)Dotzlaw, 1999Miki, 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

PXROATP-A

Miki et al, Cancer Res 2006; 66: (1). January 1, 2006

LCM/RT-PCR(C) carcinoma cells (S) stromal cells

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.

Chen Y et al., Cancer Res 2007;67:10361-10367

CTRL0.2 μM SR12813 1 μM SR12813

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

SKOV-3

D. Gupta et al. Human cancer Biology, 2008

Increased chemoresistance in ovarian carcinoma cells by PXR agonist

Systemic drug clearance Cancer cells ?

Role of CAR and PXR in intra-tumoral metabolism of irinotecan ?

Raynal C. et al. Mol. Cancer Res 2010

PXR expression in normal and cancerous human colon tissues

SW620LS174T

PXR expression level restricts SN38 chemosensibility in colon cancer cells

PXR expression level restricts SN38 chemosensibility in colon cancer cells

Intracellular metabolites profiles (HPLC

PXR expression level enhances SN38 glucuronidation in colon cancer cells

SN38-G(inactive metabolite)

SN38(cytotoxic metabolite)

PXR UGT1As

PXR increases UGT1As-mediated SN38 inactivation in colon cancer cells

SN38 SN38-G

SN38-G(inactive metabolite)

SN38(cytotoxic metabolite)

CAR/PXRUGT1A1

Pro-drug activation Cancer cells ?

Role of CAR and PXR in intra-tumoral metabolism of cyclophosphamide ?

PXR regulates ALDH1A1 gene expression and Aldefluor® activity

Hepatic cells

Cancer cells

CAR and PXR may increase cancer cell resistance to 4-OH-CPA while promoting severe toxicity

Aldefluor®-positive cells are associated with cancer initiation properties, chemotherapy-resistance and poor clinical outcome

a

Aldehyde Dehydrogenase 1 is a marker for normal and malignant human colonic stem cells and have tumor initiating properties

CD44ALDH1Nuclei

Aldefluor®-positive Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy

Aldefluor®-positive cells display higher expression of PXR and PXR target genes

PX

R

AL

DH

1A

1

AB

CG

2

CY

P3

A4

OC

T4

LG

R5

CD

26

BM

I1

NA

NO

G

PXR

Aldefluor®-positive cells display enhanced clonogenic, sphere forming activity and magnétorésistance

ALDHbrALDHlow

Nb

sp

he

re /

10

0 c

ells *

Soft-agar (1000 cells) – 3 weeks

siRNA (sibGAL / siPXR) 100nM

Cell viability (ATP content)

PXR knock-down decreases chemoresistance of Aldefluor®-positive cells

ONCO TALK - 29/01/2013

PXR knock-down decreases xenogeneic tumor recurrence

Tumor formation

Treatment response

Relapse

Folfiri: 50mg/kg 5-FU30mg/kg irinotecan

15.000 cells 3D

Tumor cells dissociation

PXR knock-down decreases Aldefluor®-positive cells enrichment and tumor initiating activity after cytotoxics treatment

FOLFIRI 1500 cellsreinjection

PXR inhibition as a new strategy for Chemoresistant and Tumor Initiating Cells (CTIC) re-sensitization to conventional therapies ?

PXR inhibition& conventional

therapy

Conventional therapy

Les xénorécepteurs CAR et PXR

Gènes CiblesGènes Cibles

Enzymes et transpoteurs majeurs de la fonction de détoxication entérohépatique

OATP2CYP3A4,5,7

CYP2B6CYP2C8,9CYP1A2

UGT1A1,6,9GSTs

SULT2A1

MDR1MRP2-4

Adduit - Hépatite fulminante- HCC

Xénobiotique

CAR/PXR

Transporteurs Métabolites CYP450 Transférases

Interactionsmédicamenteuses

Molécule thérapeutique

inactifactif

Perturbations métaboliques

Perturbationsendocriniennes

- Lipides

- Vitamine DInteractions croiséesavec d’autres voies

cellulaires

89

90

Conclusion

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

Cells and organisms are able to increase and adapt their capacity of detoxication in response to some xenobiotics and drugs

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 intra- and 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.