Cours Pascussi 2013

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 (10M)
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.