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International audience; Tumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

Purpose: Patients with metastatic colorectal cancer suffer from disease relapse mainly due to cancer stem cells (CSC). Interestingly, they have an increased level of blood progastrin, a tumor-promoting peptide essential for the self-renewal of colon CSCs, which is also a direct β-catenin/TCF4 target gene. In this study, we aimed to develop a novel targeted therapy to neutralize secreted progastrin to inhibit Wnt signaling, CSCs, and reduce relapses.Experimental Design: Antibodies (monoclonal and humanized) directed against progastrin were produced and selected for target specificity and affinity. After validation of their effectiveness on survival of colorectal cancer cell lines harboring B-RAF or K-RAS mutations, their efficacy was assessed in vitro and in vivo, alone or concomitantly with chemotherapy, on CSC self-renewal capacity, tumor recurrence, and Wnt signaling.Results: We show that anti-progastrin antibodies decrease self-renewal of CSCs both in vitro and in vivo, either alone or in combination with chemotherapy. Furthermore, migration and invasion of colorectal cancer cells are diminished; chemosensitivity is prolonged in SW620 and HT29 cells and posttreatment relapse is significantly delayed in T84 cells, xenografted nude mice. Finally, we show that the Wnt signaling activity in vitro is decreased, and, in transgenic mice developing Wnt-driven intestinal neoplasia, the tumor burden is alleviated, with an amplification of cell differentiation in the remaining tumors.Conclusions: Altogether, these data show that humanized anti-progastrin antibodies might represent a potential new treatment for K-RAS–mutated colorectal patients, for which there is a crucial unmet medical need. Clin Cancer Res; 23(17); 5267–80. ©2017 AACR.

This table presents the Top 10 most enriched KEGG pathways identified from the panel of miRNAs regulated by claudin-2, along with their enrichment score and p-value.

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This file contains all supplemental Figures, as well as legends for both Supplemental Figures and Supplemental Tables, as well as Supplemental methods and associated references

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Posttreatment recurrence of colorectal cancer, the third most lethal cancer worldwide, is often driven by a subpopulation of cancer stem cells (CSC). The tight junction (TJ) protein claudin-2 is overexpressed in human colorectal cancer, where it enhances cell proliferation, colony formation, and chemoresistance in vitro. While several of these biological processes are features of the CSC phenotype, a role for claudin-2 in the regulation of these has not been identified. Here, we report that elevated claudin-2 expression in stage II/III colorectal tumors is associated with poor recurrence-free survival following 5-fluorouracil–based chemotherapy, an outcome in which CSCs play an instrumental role. In patient-derived organoids, primary cells, and cell lines, claudin-2 promoted colorectal cancer self-renewal in vitro and in multiple mouse xenograft models. Claudin-2 enhanced self-renewal of ALDHHigh CSCs and increased their proportion in colorectal cancer cell populations, limiting their differentiation and promoting the phenotypic transition of non-CSCs toward the ALDHHigh phenotype. Next-generation sequencing in ALDHHigh cells revealed that claudin-2 regulated expression of nine miRNAs known to control stem cell signaling. Among these, miR-222-3p was instrumental for the regulation of self-renewal by claudin-2, and enhancement of this self-renewal required activation of YAP, most likely upstream from miR-222-3p. Taken together, our results indicate that overexpression of claudin-2 promotes self-renewal within colorectal cancer stem-like cells, suggesting a potential role for this protein as a therapeutic target in colorectal cancer.Significance: Claudin-2-mediated regulation of YAP activity and miR-222-3p expression drives CSC renewal in colorectal cancer, making it a potential target for therapy. Cancer Res; 78(11); 2925–38. ©2018 AACR.

Annexin II expression is not enriched in cancer stem cell populations.

Description of additional methods and procedures used in the study.

Subpopulations of cancer stem–like cells (CSC) are thought to drive tumor progression and posttreatment recurrence in multiple solid tumors. However, the mechanisms that maintain stable proportions of self-renewing CSC within heterogeneous tumors under homeostatic conditions remain poorly understood. Progastrin is a secreted peptide that exhibits tumor-forming potential in colorectal cancer, where it regulates pathways known to modulate colon CSC behaviors. In this study, we investigated the role of progastrin in regulating CSC phenotype in advanced colorectal cancer. Progastrin expression and secretion were highly enriched in colon CSC isolated from human colorectal cancer cell lines and colon tumor biopsies. Progastrin expression promoted CSC self-renewal and survival, whereas its depletion by RNA interference–mediated or antibody-mediated strategies altered the homeostatic proportions of CSC cells within heterogeneous colorectal cancer tumors. Progastrin downregulation also decreased the frequency of ALDHhigh cells, impairing their tumor-initiating potential, and inhibited the high glycolytic activity of ALDHhigh CSC to limit their self-renewal capability. Taken together, our results show how colorectal CSC maintain their tumor-initiating and self-renewal capabilities by secreting progastrin, thereby contributing to the tumor microenvironment to support malignancy. Cancer Res; 76(12); 3618–28. ©2016 AACR.

Legend for Supplemental Figures S1-S6.

Simple Summary In the present work, we describe (for the first time) the use of the transmembrane protein, CD44v6, to detect CTCs from blood samples of several patients with colorectal or breast cancer. We used CD44v6 antibodies to demonstrate that live CTCs can be specifically purified from CRC patient blood samples via magnetic bead- or FACS-based isolation techniques. Finally, we demonstrated that CD44v6-positive CTCs rarely expressed EpCam, which is currently the gold standard to enumerate CTCs, suggesting the need to use a combination of markers for a more comprehensive view of CTC heterogeneity. Abstract Circulating tumor cells (CTCs) are promising diagnostic and prognostic tools for clinical use. In several cancers, including colorectal and breast, the CTC load has been associated with a therapeutic response as well as progression-free and overall survival. However, counting and isolating CTCs remains sub-optimal because they are currently largely identified by epithelial markers such as EpCAM. New, complementary CTC surface markers are therefore urgently needed. We previously demonstrated that a splice variant of CD44, CD44 variable alternative exon 6 (CD44v6), is highly and specifically expressed by CTC cell lines derived from blood samples in colorectal cancer (CRC) patients. Two different approaches—immune detection coupled with magnetic beads and fluorescence-activated cell sorting—were optimized to purify CTCs from patient blood samples based on high expressions of CD44v6. We revealed the potential of the CD44v6 as a complementary marker to EpCAM to detect and purify CTCs in colorectal cancer blood samples. Furthermore, this marker is not restricted to colorectal cancer since CD44v6 is also expressed on CTCs from breast cancer patients. Overall, these results strongly suggest that CD44v6 could be useful to enumerate and purify CTCs from cancers of different origins, paving the way to more efficacious combined markers that encompass CTC heterogeneity.

Patients with advanced hepatocellular carcinoma (HCC) or metastatic colorectal cancer (mCRC) have a very poor prognosis due to the lack of efficient treatments. As observed in several other tumors, the effectiveness of treatments is mainly hampered by the presence of a highly tumorigenic sub-population of cancer cells called cancer stem cells (CSCs). Indeed, CSCs are resistant to chemotherapy and radiotherapy and have the ability to regenerate the tumor bulk. Hence, innovative drugs that are efficient against both bulk tumor cells and CSCs would likely improve cancer treatment. In this study, we demonstrated that GNS561, a new autophagy inhibitor that induces lysosomal cell death, showed significant activity against not only the whole tumor population but also a sub-population displaying CSC features (high ALDH activity and tumorsphere formation ability) in HCC and in liver mCRC cell lines. These results were confirmed in vivo in HCC from a DEN-induced cirrhotic rat model in which GNS561 decreased tumor growth and reduced the frequency of CSCs (CD90+CD45-). Accordingly, GNS561, which was in a global phase 1b clinical trial in liver cancers that was recently successful, offers great promise for cancer therapy by exterminating both the tumor bulk and the CSC sub-population.

OBJECTIVE Available evidence points to a role of cytochrome P450 (Cyp) drug biotransformation enzymes in central nervous system diseases, including epilepsy. Deviations in drug pharmacokinetic profiles may impact therapeutic outcomes. Here, we ask whether spontaneous recurrent seizure (SRS) activity is sufficient to modulate the expression of major Cyp enzymes in the liver and brain. METHODS Unilateral intrahippocampal (IH) kainic acid (KA) injections were used to elicit nonconvulsive status epilepticus (SE), epileptogenesis, and SRS, as monitored by video-electroencephalography. Intraperitoneal (IP) KA injection was used to trigger generalized tonic-clonic SE. KA-injected mice and sham controls were sacrificed at 24-72 hours and 1 week post-SE (IH or IP KA), and during the chronic stage (SRS; 6 weeks post-IH KA). Liver and brain tissues were processed for histology, real-time quantitative polymerase chain reaction, Western blot, or microsomal enzymatic assay. Cyp2e1, Cyp3a13, glial fibrillary acidic protein (GFAP), IBA1, xenobiotic nuclear receptors nr1i2 (PXR), nr1i3 (CAR) and nr3c1 (glucocorticoid receptor [GR]) expression was examined. Serum samples were obtained to assay corticosterone levels, a GR activator. RESULTS A significant increase of Cyp3a13 and Cyp2e1 transcript level and protein expression was found in the liver and hippocampi during SRS, as compared to control mice. In the ipsilateral hippocampus, Cyp2e1 and Cyp3a protein upregulation during SRS positively correlated to GFAP expression. GFAP+ , and not IBA1+ , cells colocalized with Cyp2e1 or Cyp3a expression. In the liver, a trend increase in Cyp3a microsomal activity was found during SRS as compared to control mice. The transcript levels of the Cyp upstream regulators GR, xenobiotic nr1i2, and nr1i3 receptors were unchanged at SRS. Corticosterone levels, a GR ligand, were increased in the blood post-SE. SIGNIFICANCE SRS modifies Cyp expression in the liver and the hippocampus. Nuclear receptors or inflammatory pathways are candidate mechanisms of Cyp regulation during seizures.

International audience; Colorectal cancer lethality usually results from post-treatment relapse in the majority of stage II-IV patients, due to the enhanced resistance of Cancer Stem Cells (CSCs). Here, we show that the nuclear receptor Pregnane X Receptor (PXR, NR1I2), behaves as a key driver of CSC-mediated tumor recurrence. First, PXR is specifically expressed in CSCs, where it drives the expression of genes involved in self-renewal and chemoresistance. Clinically, high levels of PXR correlate with poor recurrence-free survival in a cohort of >200 stage II/III colorectal cancer patients treated with chemotherapy, for whom finding biomarkers of treatment outcome is an urgent clinical need. shRNA silencing of PXR increased the chemo-sensitivity of human colon CSCs, reduced their self-renewal and tumor-initiating potential, and drastically delayed tumor recurrence in mice following chemotherapy. This study uncovers PXR as a key factor for CSC self-renewal and chemoresistance and targeting PXR thus represents a promising strategy to minimize colorectal cancer relapse by selectively sensitizing CSCs to chemotherapy.

SUPPLEMENTARY DATA are available online at http://toxsci.oxfordjournals.org/; International audience; Cytochrome P450 (CYP) expression and activity are not homogeneous in the liver lobules. Indeed, CYPs are mainly expressed and induced in centrilobular hepatocytes. The wingless-type MMTV integration site family (WNT)/β-catenin pathway was identified as a major regulator of this zonal organization. We have recently demonstrated that in primary human hepatocytes (PHHs), the expression of CYP2E1, CYP1A2, and aryl hydrocarbon receptor (AhR), but not of CYP3A4, is regulated by the WNT/β-catenin pathway in response to WNT3a, its canonical activator. Here, we investigated whether glycogen synthase kinase 3β (GSK3β) inhibitors, which mimic the action of WNT molecules, could be used in PHHs to activate the β-catenin pathway to study CYP expression. We assessed the activity of 6BIO (6-bromoindirubin-3'-oxime), CHIR99021 (6-((2-((4-(2,4-dichlorophenyl)-5-(4methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino) nicotinonitrile), and GSK3iXV (Pyridocarbazolo-cyclopentadienyl Ruthenium complex GSK3 inhibitor XV) that belong to structurally different families of GSK3β inhibitors. Using small interfering RNAs, reporter gene assays, and molecular docking predictions, we demonstrated that GSK3β inhibitors can activate the WNT/β-catenin pathway in PHHs to regulate CYP2E1 expression. We also found that 6BIO and GSK3iXV are AhR full agonists that participate, through AhR signaling, to CYP1A2 induction. Conversely, CHIR99021 is an AhR partial agonist, and a pregnane X receptor ligand and partial agonist, thus regulating CYP1A2 and CYP3A4 gene expression in a β-catenin-independent manner. In conclusion, GSK3β inhibitors can activate the WNT/β-catenin pathway in PHHs. Nevertheless, their role in CYP regulation should be analyzed with caution as these molecules can interact with xenosensors.

Nuclear receptors (NR) are emerging as key players in the central nervous system (CNS) with reported implications in physiological and pathophysiological conditions. While other NR have been studied, it is unknown whether invalidation of the pregnane xenobiotic receptor (PXR, NR1I2) corresponds to neurological modifications in the adult brain. PXR-/- C57BL/6j and wild type mice were used to investigate: i) recognition memory, motor coordination, and anxiety-like behaviors; ii) longitudinal video-electroencephalographic (EEG) recordings and frequency wave analysis; iii) neurovascular structures by histological evaluation and expression of the cerebrovascular tight junctions ZO1 and CLDN5. Absence of PXR was associated with anxiety-like behavior and recognition memory impairment in adult mice. The latter was simultaneous to an electroencephalographic signature of lower theta frequency during sleep and abnormal delta waves. Neurophysiological changes did not correspond to significant structural changes in the adult brain, expect for a localized and minor increase in the fronto-parietal neurovascular density and reduced ZO1, but not CLDN5, expression in isolated brain capillaries. Our results converge with existing evidence supporting a link between NR expression and brain physiology. Although the exact modalities remain to be elucidated, the possibility that extra-physiological modulation of PXR may constitute a pathophysiological entry point or a molecular target for brain diseases is proposed.

Posttreatment recurrence of colorectal cancer, the third most lethal cancer worldwide, is often driven by a subpopulation of cancer stem cells (CSC). The tight junction (TJ) protein claudin-2 is overexpressed in human colorectal cancer, where it enhances cell proliferation, colony formation, and chemoresistance in vitro. While several of these biological processes are features of the CSC phenotype, a role for claudin-2 in the regulation of these has not been identified. Here, we report that elevated claudin-2 expression in stage II/III colorectal tumors is associated with poor recurrence-free survival following 5-fluorouracil–based chemotherapy, an outcome in which CSCs play an instrumental role. In patient-derived organoids, primary cells, and cell lines, claudin-2 promoted colorectal cancer self-renewal in vitro and in multiple mouse xenograft models. Claudin-2 enhanced self-renewal of ALDHHigh CSCs and increased their proportion in colorectal cancer cell populations, limiting their differentiation and promoting the phenotypic transition of non-CSCs toward the ALDHHigh phenotype. Next-generation sequencing in ALDHHigh cells revealed that claudin-2 regulated expression of nine miRNAs known to control stem cell signaling. Among these, miR-222-3p was instrumental for the regulation of self-renewal by claudin-2, and enhancement of this self-renewal required activation of YAP, most likely upstream from miR-222-3p. Taken together, our results indicate that overexpression of claudin-2 promotes self-renewal within colorectal cancer stem-like cells, suggesting a potential role for this protein as a therapeutic target in colorectal cancer. Significance: Claudin-2-mediated regulation of YAP activity and miR-222-3p expression drives CSC renewal in colorectal cancer, making it a potential target for therapy. Cancer Res; 78(11); 2925–38. ©2018 AACR.

International audience; OBJECTIVE:Although counting of circulating tumour cells (CTC) has attracted a broad interest as potential markers of tumour progression and treatment response, the lack of functional characterisation of these cells had become a bottleneck in taking these observations to the clinic. Our objective was to culture these cells in order to understand them and exploit their therapeutic potential to the full.DESIGN:Here, hypothesising that some CTC potentially have cancer stem cell (CSC) phenotype, we generated several CTC lines from the blood of patients with advanced metastatic colorectal cancer (CRC) based on their self-renewal abilities. Multiple standard tests were then employed to characterise these cells.RESULTS:Our CTC lines self-renew, express CSC markers and have multilineage differentiation ability, both in vitro and in vivo. Patient-derived CTC lines are tumorigenic in subcutaneous xenografts and are also able to colonise the liver after intrasplenic injection. RNA sequencing analyses strikingly demonstrate that drug metabolising pathways represent the most upregulated feature among CTC lines in comparison with primary CRC cells grown under similar conditions. This result is corroborated by the high resistance of the CTC lines to conventional cytotoxic compounds.CONCLUSIONS:Taken together, our results directly demonstrate the existence of patient-derived colorectal CTCs that bear all the functional attributes of CSCs. The CTC culture model described here is simple and takes

Purpose: Patients with metastatic colorectal cancer suffer from disease relapse mainly due to cancer stem cells (CSC). Interestingly, they have an increased level of blood progastrin, a tumor-promoting peptide essential for the self-renewal of colon CSCs, which is also a direct β-catenin/TCF4 target gene. In this study, we aimed to develop a novel targeted therapy to neutralize secreted progastrin to inhibit Wnt signaling, CSCs, and reduce relapses. Experimental Design: Antibodies (monoclonal and humanized) directed against progastrin were produced and selected for target specificity and affinity. After validation of their effectiveness on survival of colorectal cancer cell lines harboring B-RAF or K-RAS mutations, their efficacy was assessed in vitro and in vivo, alone or concomitantly with chemotherapy, on CSC self-renewal capacity, tumor recurrence, and Wnt signaling. Results: We show that anti-progastrin antibodies decrease self-renewal of CSCs both in vitro and in vivo, either alone or in combination with chemotherapy. Furthermore, migration and invasion of colorectal cancer cells are diminished; chemosensitivity is prolonged in SW620 and HT29 cells and posttreatment relapse is significantly delayed in T84 cells, xenografted nude mice. Finally, we show that the Wnt signaling activity in vitro is decreased, and, in transgenic mice developing Wnt-driven intestinal neoplasia, the tumor burden is alleviated, with an amplification of cell differentiation in the remaining tumors. Conclusions: Altogether, these data show that humanized anti-progastrin antibodies might represent a potential new treatment for K-RAS–mutated colorectal patients, for which there is a crucial unmet medical need. Clin Cancer Res; 23(17); 5267–80. ©2017 AACR.

International audience; The Constitutive Androstane Receptor (CAR, NR1I3) has been newly described as a regulator of energy metabolism. A relevant number of studies using animal models of obesity suggest that CAR activation could be beneficial on the metabolic balance. However, this remains controversial and the underlying mechanisms are still unknown. This work aimed to investigate the effect of CAR activation on hepatic energy metabolism during physiological conditions, i.e. in mouse models not subjected to metabolic/nutritional stress. Gene expression profiling in the liver of CAR knockout and control mice on chow diet and treated with a CAR agonist highlighted CAR-mediated up-regulations of lipogenic genes, concomitant with neutral lipid accumulation. A strong CAR-mediated up-regulation of the patatin-like phospholipase domain-containing protein 3 (Pnpla3) was demonstrated. Pnpla3 is a gene whose polymorphism is associated with the pathogenesis of nonalcoholic fatty liver disease (NAFLD) development. This observation was confirmed in human hepatocytes treated with the antiepileptic drug and CAR activator, phenobarbital and in immortalized human hepatocytes treated with CITCO. Studying the molecular mechanisms controlling Pnpla3 gene expression, we demonstrated that CAR does not act by a direct regulation of Pnpla3 transcription or via the Liver X Receptor but may rather involve the transcription factor Carbohydrate Responsive Element-binding protein. These data provide new insights into the regulation by CAR of glycolytic and lipogenic genes and on pathogenesis of steatosis. This also raises the question concerning the impact of drugs and environmental contaminants in lipid-associated metabolic diseases.

Subpopulations of cancer stem–like cells (CSC) are thought to drive tumor progression and posttreatment recurrence in multiple solid tumors. However, the mechanisms that maintain stable proportions of self-renewing CSC within heterogeneous tumors under homeostatic conditions remain poorly understood. Progastrin is a secreted peptide that exhibits tumor-forming potential in colorectal cancer, where it regulates pathways known to modulate colon CSC behaviors. In this study, we investigated the role of progastrin in regulating CSC phenotype in advanced colorectal cancer. Progastrin expression and secretion were highly enriched in colon CSC isolated from human colorectal cancer cell lines and colon tumor biopsies. Progastrin expression promoted CSC self-renewal and survival, whereas its depletion by RNA interference–mediated or antibody-mediated strategies altered the homeostatic proportions of CSC cells within heterogeneous colorectal cancer tumors. Progastrin downregulation also decreased the frequency of ALDHhigh cells, impairing their tumor-initiating potential, and inhibited the high glycolytic activity of ALDHhigh CSC to limit their self-renewal capability. Taken together, our results show how colorectal CSC maintain their tumor-initiating and self-renewal capabilities by secreting progastrin, thereby contributing to the tumor microenvironment to support malignancy. Cancer Res; 76(12); 3618–28. ©2016 AACR.

In vitro production of human hepatocytes is of primary importance in basic research, pharmacotoxicology and biotherapy of liver diseases. We have developed a protocol of differentiation of human embryonic stem cells (ES) towards hepatocyte-like cells (ES-Hep). Using a set of human adult markers including CAAT/enhancer binding protein (C/EBPalpha), hepatocyte nuclear factor 4/7 ratio (HNF4alpha1/HNF4alpha7), cytochrome P450 7A1 (CYP7A1), CYP3A4 and constitutive androstane receptor (CAR), and fetal markers including alpha-fetoprotein, CYP3A7 and glutathione S-transferase P1, we analyzed the expression of a panel of 41 genes in ES-Hep comparatively with human adult primary hepatocytes, adult and fetal liver. The data revealed that after 21 days of differentiation, ES-Hep are representative of fetal hepatocytes at less than 20 weeks of gestation. The glucocorticoid receptor pathway was functional in ES-Hep. Extending protocols of differentiation to 4 weeks did not improve cell maturation. When compared with hepatocyte-like cells derived from adult liver non parenchymal epithelial (NPE) cells (NPE-Hep), ES-Hep expressed several adult and fetal liver makers at much greater levels (at least one order of magnitude), consistent with greater expression of liver-enriched transcription factors Forkhead box A2, C/EBPalpha, HNF4alpha and HNF6. It therefore seems that ES-Hep reach a better level of differentiation than NPE-Hep and that these cells use different lineage pathways towards the hepatic phenotype. Finally we showed that lentivirus-mediated expression of xenoreceptor CAR in ES-Hep induced the expression of several detoxification genes including CYP2B6, CYP2C9, CYP3A4, UDP-glycosyltransferase 1A1, solute carriers 21A6, as well as biotransformation of midazolam, a CYP3A4-specific substrate. Electronic supplementary material The online version of this article (doi:10.1007/s12015-010-9225-3) contains supplementary material, which is available to authorized users.

International audience; BACKGROUND/AIMS: The aim of this study was to identify human liver proteins that are associated with different stages of liver development. METHODS: We collected liver samples from 14 fetuses between 14 and 41 weeks of development, one child and four adults. Proteins which exhibited consistent and significant variations during development by two-dimensional differential in gel electrophoresis (2D-DIGE) were subjected to peptide mass fingerprint analysis by MALDI-TOF mass spectrometry. Real-time PCR analysis confirmed, at the transcriptional level, the data obtained by the proteomic approach. RESULTS: Among a total of 80 protein spots showing differential expression, we identified 42 different proteins or polypeptide chains, of which 26 were upregulated and 16 downregulated in developing in comparison to adult liver. These proteins could be classified in specific groups according to their function. By comparing their temporal expression profiles, we identified protein groups that were associated with different developmental stages of human fetal liver and suggest that the changes in protein expression observed during the 20- to 36-week time window play a pivotal role in liver development. CONCLUSIONS: The identification of these proteins may represent good markers of human liver and stem cells differentiation.

The widely prescribed lipid-lowering statins are considered to be relatively safe drugs. However, the risk of severe myopathy and drug interactions as a consequence of statin therapy provides a challenge for development of novel cholesterol-lowering agents, targeting enzymes other than HMG-CoA reductase. The novel pyridylethanol-(phenylethyl)amine derivative, (2-((3,4-dichlorophenethyl)(propyl)-amino)-1-(pyridin-3-yl)ethanol (LK-935), blocking lanosterol 14alpha-demethylase, was demonstrated to efficiently reduce cholesterol biosynthesis. The drug interaction potential of LK-935 was investigated and compared with that of atorvastatin and rosuvastatin in primary human hepatocytes. Clear evidence was provided for the induction of CYP3A4 by LK-935. LK-935 was proved to be a potent human pregnane X receptor (hPXR) activator as a prerequisite for the transcriptional activation of CYP3A4 gene; however, the rapid metabolism of LK-935 in primary hepatocytes prevented maximal CYP3A4 induction. Therefore, the induction of CYP3A4 by LK-935 may be prone to mild or negligible drug interactions. However, because CYP3A4 and also CYP2C9 play a significant role in LK-935 metabolism, the inhibition of these cytochromes P450 by coadministered drugs may lead to some increase in the LK-935 concentration required for the potent induction of CYP3A4. Rosuvastatin was found to increase human constitutive androstane receptor (hCAR)-mediated transcription of CYP3A4, CYP2C9, and CYP2B6 genes, predicting the consequent potential for drug interactions with several coadministered drugs. Activation of hCAR and hPXR by atorvastatin and the subsequent induction of not only CYP2B6 and CYP3A4 but also of CYP2C9 present an additional target by which atorvastatin, a widely used cholesterol-lowering drug, can modify the kinetics of numerous drugs.

Decreased drug metabolism, hyperbilirubinemia and intrahepatic cholestasis are frequently observed during inflammation. Additionally, it has long been appreciated that exposure to drug metabolism-inducing xenobiotics can impair immune function. The nuclear receptor CAR (constitutive androstane receptor or NR1I3) and PXR (pregnane X receptor, NR1I2) control phase I (cytochrome P450 2B and 3A), phase II (GSTA, UGT1A1), and transporter (MDR1, SLC21A6, MRP2) genes involved in drugs metabolism, bile acids and bilirubin clearance in response to xenobiotics. It is well known that inflammation, through the activation of NF-kappaB pathway, leads to a decrease of CAR, PXR and RXRalpha expression and the expression of their target genes. In addition, a new study reveals the mutual repression between PXR and NF-kappaB signaling pathways, providing a molecular mechanism linking xenobiotic metabolism and inflammation.

International audience; The expression of many genes involved in xenobiotic/drug metabolism and transport is regulated by at least three nuclear receptors or xenosensors: aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR). These receptors establish crosstalk with other nuclear receptors or transcription factors controlling signaling pathways that regulate the homeostasis of bile acids, lipids, glucose, inflammation, vitamins, hormones, and others. These crosstalks are expected to modify profoundly our vision of xenobiotic/drug disposition and toxicity. They provide molecular mechanisms to explain how physiopathological stimuli affect xenobiotic/drug disposition, and how xenobiotics/drugs may affect physiological functions and generate toxic responses. In addition, the possibility that xenosensors may control other signaling pathways opens the way to new pharmacological opportunities.

Objective Although counting of circulating tumour cells (CTC) has attracted a broad interest as potential markers of tumour progression and treatment response, the lack of functional characterisation of these cells had become a bottleneck in taking these observations to the clinic. Our objective was to culture these cells in order to understand them and exploit their therapeutic potential to the full. Design Here, hypothesising that some CTC potentially have cancer stem cell (CSC) phenotype, we generated several CTC lines from the blood of patients with advanced metastatic colorectal cancer (CRC) based on their self-renewal abilities. Multiple standard tests were then employed to characterise these cells. Results Our CTC lines self-renew, express CSC markers and have multilineage differentiation ability, both in vitro and in vivo. Patient-derived CTC lines are tumorigenic in subcutaneous xenografts and are also able to colonise the liver after intrasplenic injection. RNA sequencing analyses strikingly demonstrate that drug metabolising pathways represent the most upregulated feature among CTC lines in comparison with primary CRC cells grown under similar conditions. This result is corroborated by the high resistance of the CTC lines to conventional cytotoxic compounds. Conclusions Taken together, our results directly demonstrate the existence of patient-derived colorectal CTCs that bear all the functional attributes of CSCs. The CTC culture model described here is simple and takes

International audience; Nuclear receptors (NRs) are a group of transcription factors emerging as players in normal and pathological CNS development. Clinically, an association between the constitutive androstane NR (CAR) and cognitive impairment was proposed, however never experimentally investigated. We wished to test the hypothesis that the impact of CAR on neurophysiology and behavior is underlined by cerebrovascular-neuronal modifications. We have used CAR(-/-) C57BL/6 and wild type mice and performed a battery of behavioral tests (recognition, memory, motor coordination, learning and anxiety) as well as longitudinal video-electroencephalographic recordings (EEG). Brain cell morphology was assessed using 2-photon or electron microscopy and fluorescent immunohistochemistry. We observed recognition memory impairment and increased anxiety-like behavior in CAR(-/-) mice, while loco motor activity was not affected. Concomitantly to memory deficits, EEG monitoring revealed a decrease in 3.5-7 Hz waves during the awake/exploration and sleep periods. Behavioral and EEG abnormalities in CAR(-/-) mice mirrored structural changes, including tortuous fronto-parietal penetrating vessels. At the cellular level we found reduced ZO-1, but not CLDN5, tight junction protein expression in cortical and hippocampal isolated microvessel preparations. Interestingly, the neurotoxin kainic acid, when injected peripherally, provoked a rapid onset of generalized convulsions in CAR(-/-) as compared to WT mice, supporting the hypothesis of vascular permeability. The morphological phenotype of CAR(-/-) mice also included some modifications of GFAP/IBA1 glial cells in the parenchymal or adjacent to collagen-IV+ or FITC+ microvessels. Neuronal defects were also observed including increased cortical NEUN+ cell density, hippocampal granule cell dispersion and increased NPY immunoreactivity in the CA1 region in CAR(-/-) mice. The latter may contribute to the in vivo phenotype. Our results indicate that behavioral and electroencephalographic changes in adult CAR(-/-) mice are concomitant to discrete developmental or structural brain defects. The latter could increase the vulnerability to neurotoxins. The possibility that interfering with nuclear receptors during development could contribute to adulthood brain changes is proposed.

Constitutive androstane receptor (CAR; NR1I3) controls the metabolism and elimination of endogenous and exogenous toxic compounds by up-regulating a battery of genes. In this work, we analyzed the expression of human CAR (hCAR) in normal liver during development and in hepatocellular carcinoma (HCC) and investigated the effect of hepatocyte nuclear factor 4alpha isoforms (HNF4alpha1 and HNF4alpha7) on the hCAR gene promoter. By performing functional analysis of hCAR 5'-deletions including mutants, chromatin immunoprecipitation in human hepatocytes, electromobility shift and cotransfection assays, we identified a functional and species-conserved HNF4alpha response element (DR1: ccAGGCCTtTGCCCTga) at nucleotide -144. Both HNF4alpha isoforms bind to this element with similar affinity. However, HNF4alpha1 strongly enhanced hCAR promoter activity whereas HNF4alpha7 was a poor activator and acted as a repressor of HNF4alpha1-mediated transactivation of the hCAR promoter. PGC1alpha stimulated both HNF4alpha1-mediated and HNF4alpha7-mediated hCAR transactivation to the same extent, whereas SRC1 exhibited a marked specificity for HNF4alpha1. Transduction of human hepatocytes by HNF4alpha7-expressing lentivirus confirmed this finding. In addition, we observed a positive correlation between CAR and HNF4alpha1 mRNA levels in human liver samples during development, and an inverse correlation between CAR and HNF4alpha7 mRNA levels in HCC. These observations suggest that HNF4alpha1 positively regulates hCAR expression in normal developing and adult livers, whereas HNF4alpha7 represses hCAR gene expression in HCC.

We report here the first association between vitamin D–resistant rickets, alopecia, and type 1 diabetes in a child with compound heterozygous mutations in the VDR gene. Transfection studies suggest dissociated effects of VDR gene mutations on the regulation of genes involved in vitamin D metabolism and dendritic cell maturation. Introduction: Whereas vitamin D may play a role in the immune tolerance process, no patient has been reported to associate hereditary vitamin D–resistant rickets (HVDRR) and an autoimmune disease, and no attempt has been made to delineate the outcome of mutations of the vitamin D receptor (VDR) on the transcription of genes controlling immune tolerance. Materials and Methods: The VDR gene was analyzed in a child with vitamin D–resistant rickets, total alopecia, and early childhood–onset type 1 diabetes. Patient's fibroblasts and COS-7 cells transfected with wildtype or mutant VDRs were studied for ligand-binding capacity, transactivation activity using two gene promoters [CYP-24, a classical 1,25(OH)2D3-responsive gene, and relB, a critical NF-κB component for regulation of dendritic cell differentiation], VDR-RXR heterodimers association to CYP 24 VDREs by gel mobility shift assays, and co-activator binding by Glutathione-S-transferase pull-down assays. Results: Two novel compound heterozygous mutations (L263R and R391S) were identified in the VDR ligand-binding domain in this child. Both mutations significantly impaired VDR ligand-binding capacity but had dissociated effects on CYP-24 and RelB promoter responses to vitamin D. CYP 24 response binding to SRC-1 and RXR-heterodimer binding to CYP24 VDREs were abolished in L263R mutants but normal or partially altered in R391S mutants. In the opposite, RelB responses to vitamin D were close to normal in L263R mutants but abolished in R391S mutants. Conclusions: We report the first clinical association between HVDRR, total alopecia, and early childhood–onset type 1 diabetes. Mutations in the VDR ligand-binding domain may hamper the 1,25(OH)2D3–mediated relB responses, an effect that depends on the site of the VDR mutation and cannot be anticipated from VDR ligand-binding ability or CYP-24 response. Based on these results, we propose to survey the immune function in patients with HVDRR, including those with moderate features of rickets.

International audience; Pregnane X receptor (PXR, NR1I2) is activated by various chemically unrelated compounds, including environmental pollutants and drugs. We proceeded here to in vitro screening of 28 pesticides with a new reporter system that detects human pregnane X receptor (hPXR) activators. The cell line was obtained by a two-step stable transfection of cervical cancer HeLa cells. The first transfected cell line, HG5LN, contained an integrated luciferase reporter gene under the control of a GAL4 yeast transcription factor-binding site. The second cell line HGPXR was derived from HG5LN and stably expressed hPXR ligand-binding domain fused to GAL4 DNA-binding domain (DBD). The HG5LN cells were used as a control to detect nonspecific activities. Pesticides from various chemical classes were demonstrated, for the first time, to be hPXR activators: (1) herbicides: pretilachlor, metolachlor, and alachlor chloracetanilides, oxadiazon oxiconazole, and isoproturon urea; (2) fungicides: bupirimate and fenarimol pyrimidines, propiconazole, fenbuconazole, prochloraz conazoles, and imazalil triazole; and (3) insecticides: toxaphene organochlorine, permethrin pyrethroid, fipronil pyrazole, and diflubenzuron urea. Pretilachlor, metolachlor, bupirimate, and oxadiazon had an affinity for hPXR equal to or greater than the positive control rifampicin. Some of the newly identified hPXR activators were also checked for their ability to induce cytochrome P450 3A4 expression in a primary culture of human hepatocytes. HGPXR, with HG5LN as a reference, was grafted onto nude mice to assess compound bioavailability through in vivo quantification of hPXR activation. Altogether, our data indicate that HGPXR cells are an efficient tool for identifying hPXR ligands and establishing pesticides as hPXR activators.

Vitamin D controls calcium homeostasis and the development and maintenance of bones through vitamin D receptor activation. Prolonged therapy with rifampicin or phenobarbital has been shown to cause vitamin D deficiency or osteomalacia, particularly in patients with marginal vitamin D stores. However, the molecular mechanism of this process is unknown. Here we show that these drugs lead to the upregulation of 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24) gene expression through the activation of the nuclear receptor pregnane X receptor (PXR; NR1I2). CYP24 is a mitochondrial enzyme responsible for inactivating vitamin D metabolites. CYP24 mRNA is upregulated in vivo in mice by pregnenolone 16alpha-carbonitrile and dexamethasone, 2 murine PXR agonists, and in vitro in human hepatocytes by rifampicin and hyperforin, 2 human PXR agonists. Moreover, rifampicin increased 24-hydroxylase activity in these cells, while, in vivo in mice, pregnenolone 16alpha-carbonitrile increased the plasma concentration of 24,25-dihydroxyvitamin D(3). Transfection of PXR in human embryonic kidney cells resulted in rifampicin-mediated induction of CYP24 mRNA. Analysis of the human CYP24 promoter showed that PXR transactivates the sequence between -326 and -142. We demonstrated that PXR binds to and transactivates the 2 proximal vitamin D-responsive elements of the human CYP24 promoter. These data suggest that xenobiotics and drugs can modulate CYP24 gene expression and alter vitamin D(3) hormonal activity and calcium homeostasis through the activation of PXR.

Humans are chronically exposed to multiple exogenous substances, including environmental pollutants, drugs and dietary components. Many of these compounds are suspected to impact human health, and their combination in complex mixtures could exacerbate their harmful effects. Here we demonstrate that a pharmaceutical oestrogen and a persistent organochlorine pesticide, both exhibiting low efficacy when studied separately, cooperatively bind to the pregnane X receptor, leading to synergistic activation. Biophysical analysis shows that each ligand enhances the binding affinity of the other, so the binary mixture induces a substantial biological response at doses at which each chemical individually is inactive. High-resolution crystal structures reveal the structural basis for the observed cooperativity. Our results suggest that the formation of ‘supramolecular ligands' within the ligand-binding pocket of nuclear receptors contributes to the synergistic toxic effect of chemical mixtures, which may have broad implications for the fields of endocrine disruption, toxicology and chemical risk assessment., Endocrine-disrupting chemicals act on nuclear hormone receptors, such as PXR. Here, Delfosse et al. show how two such chemicals interact with each other in the PXR ligand-binding pocket, forming a so-called supramolecular ligand that is a more potent PXR activator than each of the two chemicals alone.

Background: In spite of wide application of sorafenib for advanced hepatocellular carcinoma (HCC) treatment, and systemic chemotherapy cocktails (5-fluorouracil, irinotecan, and oxaliplatin) for metastatic colorectal cancer, the prognostic for both cancers remains poor. In recent years, highly tumorigenic sub-populations of cancer cells named Cancer Stem Cells (CSCs) have been claimed as responsible of some tumor recurrences. Indeed, CSCs are resistant to chemotherapy, and they have the ability to regenerate all the tumor bulk with its heterogeneous cell type populations. For this reason, new drugs with original mechanism of action which target CSC properties would likely improve cancer treatment Material and methods: Antitumor activity of GNS561 was tested on a panel of cancer cell lines and primary tumors. GNS561 impact on CSCs subpopulation in patient derived cells from colorectal hepatic metastatic tumors was assessed by flow cytometry (ALDH activity). In HCC, the effect of this drug was evaluated by sphere formation assay as readout to estimate CSC survival. Tolerance and plasma and liver pharmacokinetic were evaluated after single and repeated dosing in mice and rats. In vivo GNS561 activity was tested in orthotopic mouse model Results: GNS561 demonstrated multiple cellular effects such as inhibition of autophagy, induction of apoptosis and cell cycle modulation. It showed antitumor activity against several human cancer cell lines. Furthermore, GNS561 was effective against a panel of HCC tumors even from patients harboring sorafenib resistance. GNS561 showed nonetheless an original dose-response cytotoxic activity against the whole tumor populations but also against a subpopulation displaying high ALDH activity in three CRC patient-derived cell lines established from fresh liver metastasis biopsies. Consequently, on the same model this compound induced a striking decrease of sphere formation. In HCC cell lines GNS561 was active on both whole populations (mean EC50 2µM) and subpopulations displaying CSC features (Epcam high). In addition, in the opposite of sorafenib, GNS561 decreases the HCC cell capacity to form spheroids. In mouse, GNS561 was found well tolerated and highly selectively trapped in the liver (exposure ratio liver/plasma about 170 animals). In HCC PDX mouse model, tumor growth was significantly reduced by GNS561 with a dose-response manner, this tumor regression was associated with AFP level decreases by 72% with GNS561 (p=0.002) and 54% with sorafenib (p=0.046) compared to control Conclusions: GNS561 is a liver selective drug which offers great promise for HCC and liver metastatic tumors treatment. By simultaneously targeting the cancer stem cell subpopulation and tumor bulk, both cell heterogeneity, plasticity and recurrences could be overcome at least in colorectal cancer and HCC. GNS 561 is now aimed to further reach clinical development in patients in 2017 Note: This abstract was not presented at the meeting. Citation Format: Firas Bassissi, Elena Patricia Gifu, Sonia Brun, Jerome Courcambeck, Antoine Beret, Jean Marc Pascussi, Julie Pannequin, Eric Raymond, Philippe Halfon, Philippe Merle, Claude Caron de Fromentel. GNS561 is a new quinoline derivative with high efficacy on cancer stem cells from colorectal liver metastasis and hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1914. doi:10.1158/1538-7445.AM2017-1914

P450 metabolic enzymes are expressed in the human and rodent brain. Recent data support their involvement in the pathophysiology of epilepsy. However, the determinants of metabolic enzyme expression in the epileptic brain are unclear. We tested the hypothesis that status epilepticus (SE) or exposure to phenytoin or phenobarbital affects brain expression of the metabolic enzyme CYP2E1. SE was induced in C57BL/6J mice by systemic kainic acid. Brain CYP2E1 expression was evaluated 18-24h after severe SE by immunohistochemistry. Co-localization with neuronal nuclei (NEUN), glial fibrillary acidic protein (GFAP) and CD31 was determined by confocal microscopy. The effect of phenytoin, carbamazepine and phenobarbital on CYP2E1 expression was evaluated in vivo or by using organotypic hippocampal cultures in vitro. CYP2E1 expression was investigated in brain resections from a cohort of drug-resistant epileptic brain resections and human endothelial cultures (EPI-EC). Immunohistochemistry showed an increase of CYP2E1 expression limited to hippocampal CA2/3 and hilar neurons after severe SE in mice. CYP2E1 expression was also observed at the astrocyte-vascular interface. Analysis of human brain specimens revealed CYP2E1 expression in neurons and vascular endothelial cells (EC). CYP2E1 was expressed in cultured human EC and over-expressed by EPI-EC. When analyzing the effect of drug exposure on CYP2E1 expression we found that, in vivo or in vitro, ethanol increased CYP2E1 levels in the brain and liver. Treatment with phenytoin induced localized CYP2E1 expression in the brain whereas no significant effects were exerted by carbamazepine or phenobarbital. Our data indicate that the effect of acute SE on brain CYP2E1 expression is localized and cell specific. Exposure to selected anti-epileptic drugs could play a role in determining CYP2E1 brain expression. Additional investigation is required to fully reproduce the culprits of P450 enzyme expression as observed in the human epileptic brain.

Although CYP3A induction by dexamethasone has been extensively documented, its mechanism is still unclear because both the role of the glucocorticoid receptor and the ability of dexamethasone to activate the human pregnane X receptor have been questioned. In an attempt to resolve this problem, we investigated the response of CYP3A4 to dexamethasone (10 nm-100 microm) in primary human hepatocytes and HepG2 cells, using a variety of methods: kinetic analysis of CYP3A4 and tyrosine aminotransferase expression, effects of RU486 and cycloheximide, ligand binding assay, cotransfection of HepG2 cells with CYP3A4 reporter gene constructs and vectors expressing the glucocorticoid receptor, pregnane X receptor or constitutively activated receptor. In contrast to rifampicin (monophasic induction), dexamethasone produces a biphasic induction of CYP3A4 mRNA consisting of a low-dexamethasone component (nmol concentrations) of low amplitude (factor of 3-4) followed by a high-dexamethasone component (supramicromolar concentrations) of high amplitude (factor of 15-30). We show that the low-dexamethasone component results from the glucocorticoid receptor-mediated expression of pregnane X receptor and/or constitutively activated receptor which, in turn, are able to transactivate CYP3A4 in a xenobiotic-independent manner. At supramicromolar concentrations (>10 microm), dexamethasone binds to and activates pregnane X receptor thus producing the high-dexamethasone component of CYP3A4 induction. We conclude that, in contrast to the other xenobiotic inducers of CYP3A4, glucocorticoids play a dual role in CYP3A4 expression, first by controlling the expression of PXR and CAR under physiological conditions (submicromolar concentrations) through the classical glucocorticoid receptor pathway, and second by activating the pregnane X receptor under bolus or stress conditions (supramicromolar concentrations).

International audience; Background: We previously showed that blood serum induced cytochrome P450 1A1 (CYP1A1) monooxygenase expression in vitro.Objective: Our purpose was (i) to identify the molecular mechanism involved and (ii) to characterize the inducer compound(s) in serum involved at least in part.Methods: Serum was fractionated on hydrophobic columns. PPARα involvement was demonstrated by gene reporter assays, DNA mutagenesis and EMSA. Gene expression was evaluated by qRT-PCR. Serum samples were analyzed using HS-SPME-GC-MS.Results: The inductive effect of serum did not depend on the AhR pathway and was enhanced by cotransfection of PPARα cDNA. Mutations in the PPAR response elements of the CYP1A1 gene promoter suppressed this effect. One of the PPRE sites appeared highly specific for human PPARα, an unreported PPRE property. A link was found between CYP1A1 inducibility and serum hydrophobic compounds. Characterization of sera showed that hexanal, a metabolite produced by peroxidation of linoleic acid, was involved in CYP1A1 induction by serum, possibly along with other serum entities.Conclusion: We demonstrate that serum induces CYP1A1 via the PPARα pathway and that hexanal is one of the serum inducers. The two PPRE sites within the CYP1A1 promoter are functional and one of them is specific for PPARα.

The mycotoxin zearalenone (ZEN) is found worldwide as a contaminant in cereals and grains. ZEN subchronic and chronic toxicities are dominated by reproductive disorders in different mammalian species which have made ZEN established mammalian endocrine disrupter. Over the last 30 years of ZEN biotransformation study, the toxin was thought to undergo reductive metabolism only, with the generation in several species of α- and β-isomers of zearalenol. However, recent investigations have noticed that the mycoestrogen is prone to oxidative metabolism leading to hydroxylation of ZEN though the involvement of different cytochromes P450 (CYPs) isoforms. The aim of the present study was to further explore the effect of ZEN on regulation of some CYPs using primary cultures of human hepatocytes. For this aim, using real time RT-PCR, we monitored in a first time, the effect of ZEN on mRNA levels of pregnane X receptor (PXR), constitutive androstane receptor (CAR) and aryl hydrocarbon receptor (AhR), nuclear receptors known to be involved in the regulation of some CYPs. In a second time, we looked for ZEN effect on expression of PXR, CAR and AhR corresponding phase I target genes (CYP3A4, CYP3A5, CYP2B6, CYP2C9, CYP1A1 and CYP1A2). Finally, we realised the luciferase assay in HepG2 treated with the toxin and transiently transfected with p-CYP3A4-Luc in the presence of a hPXR vector or transfected with p-CYPA1-Luc.Our results clearly showed that ZEN activated human PXR, CAR and AhR mRNA levels in addition to some of their phase I target genes mainly CYP3A4, CYP2B6 and CYP1A1 and at lesser extent CYP3A5 and CYP2C9 at ZEN concentrations as low as 0.1 μM.

The pregnane X receptors (PXRs) and the constitutive androstane receptor (CAR) were initially isolated as nuclear receptors regulating xenobiotic metabolism and elimination, alleviating chemical insults. However, recent works suggest that these xenoreceptors play an endobiotic role in modulating hepatic lipid metabolism. In this study, we show that CAR activators]phenobarbital and 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime] induce the lipogenic gene thyroid hormone-responsive spot 14 protein (THRSP) (or Spot14, S14) expression in human hepatocytes. In addition, we report that treatment of wild-type mice with mCAR activators (phenobarbital and 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene) efficiently increases thrsp expression, in contrast to CAR null mice. We demonstrate that CAR directly transactivates THRSP promoter through the direct repeat with 4-bp spacer thyroid hormone and PXR response element. Deletion or point mutations within this PXR response element led to a drastic inhibition of CAR-mediated THRSP transactivation. Gel-shift analysis revealed that the CAR/retinoid X receptor complex binds to this element. In conclusion, our results indicate that THRSP gene is a CAR and PXR target gene. Because THRSP expression correlates with lipogenesis and insulin sensitivity, our data suggest that CAR and/or PXR activating drugs and xenobiotics may promote aberrant hepatic de novo lipogenesis leading potentially to fatty liver diseases and insulin resistance.

The pregnane X receptor (PXR) initially isolated as a nuclear receptor regulating xenobiotic and drug metabolism and elimination, seems to play an endobiotic role by affecting lipid homeostasis. In mice, PXR affects lipid homeostasis and increases hepatic deposit of triglycerides. In this study, we show that, in human hepatocyte, PXR activation induces an increase of de novo lipogenesis through the up-regulation of S14. S14 was first identified as a thyroid-responsive gene and is known to transduce hormone-related and nutrient-related signals to genes involved in lipogenesis through a molecular mechanism not yet elucidated. We demonstrate that S14 is a novel transcriptional target of PXR. In addition, we report an increase of fatty acid synthase (FASN) and adenosine triphosphate citrate lyase genes expression after PXR activation in human hepatocyte, leading to an increase of fatty acids accumulation and de novo lipogenesis. RNA interference of the expression of S14 proportionally decreases the FASN induction, whereas S14 overexpression in human hepatic cells provokes an increase of fatty acids accumulation and lipogenesis. These results demonstrate for the first time that xenobiotic or drug-activated PXR promote aberrant hepatic de novo lipogenesis via activation of the nonclassical S14 pathway. In addition, these data suggest that the up-regulation of S14 by PXR may promote aberrant hepatic lipogenesis and hepatic steatosis in human hepatocytes.

Di-(2-ethylhexyl)-phthalate (DEHP), a widely used plasticizer, is detected in consumer's body fluids. Contamination occurs through environmental and food chain sources. In mouse liver, DEHP activates the peroxisome proliferator-activated receptor alpha (PPARalpha) and regulates the expression of its target genes. Several in vitro investigations support the simultaneous recruitment of additional nuclear receptor pathways. We investigated, in vivo, the hepatic impact of low doses of DEHP on PPARalpha activation, and the putative activation of additional signalling pathways. Wild-type and PPARalpha-deficient mice were exposed to different doses of DEHP. Gene expression profiling delineated the role of PPARalpha and revealed a PPARalpha-independent regulation of several prototypic constitutive androstane receptor (CAR) target genes. Thus, we developed an original hepatic cell line expressing CAR to investigate its activation by DEHP. By means of a pharmacological inhibitor or CAR-targeting shRNAs, we established that CAR is required for the effect of DEHP on Cyp2b10, a recognized CAR target gene. Moreover, DEHP dose-dependently induced CYP2B6 in human primary hepatocyte cultures. This finding demonstrates that CAR also represents a transcriptional regulator sensitive to phthalates. CAR-mediated effects of DEHP provide a new rationale for most endpoints of phthalates toxicity described previously, including endocrine disruption, hepatocarcinogenesis and the metabolic syndrome.

Decreased drug metabolism, hyperbilirubinemia and intrahepatic cholestasis are frequently observed during inflammation. Additionally, it has long been appreciated that exposure to drug metabolism-inducing xenobiotics can impair immune function. The nuclear receptor CAR (constitutive androstane receptor or NR1I3) and PXR (pregnane X receptor, NR1I2) control phase I (cytochrome P450 2B and 3A), phase II (GSTA, UGT1A1), and transporter (MDR1, SLC21A6, MRP2) genes involved in drugs metabolism, bile acids and bilirubin clearance in response to xenobiotics. It is well known that inflammation, through the activation of NF-kappaB pathway, leads to a decrease of CAR, PXR and RXRalpha expression and the expression of their target genes. In addition, a new study reveals the mutual repression between PXR and NF-kappaB signaling pathways, providing a molecular mechanism linking xenobiotic metabolism and inflammation.

It has long been demonstrated that the level of cholesterol in cells regulatesthe cholesterol biosynthesis through SREBF transcription factors, butlately it has been shown that other factors are also important. To study the system we employed Bayesian network inference and combined it with mathematical modeling and simulation. We constructed a mathematical model of cholesterol biosynthesis and studied its properties through simulation. We measured transcriptional changes of cholesterogenic genes using the Steroltalkmicroarray and treated human hepatocyte samples. We employed Bayesian inference to identify gene-to-gene interactions from both microarray measurements and simulated data. The inferred networks show that the expression of cholesterogenic genes can be predicted from the expression of 4 key genes, one of them being SREBF2. Networks also indicate a strong interaction between SREBF2 and CYP51A1, but not between SREBF2 and HMGCR, the rate-limiting enzyme of cholesterol biosynthesis. The expression of HMGCR seems to be regulated by other factor(s). Computer simulations of the mathematical model of cholesterol biosynthesis exposed that a large number of perturbations of the system is critical for identification of gene-to-gene interactions, and that differences between human individuals (biological variability) and measurement noise (technical variability) pose a serious problem for their automatic inference from DNA microarray data.

Hepatitis C virus-positive serum (HCVser, genotypes 1a to 3a) or HCV cell culture (JFH1/HCVcc) infection of primary normal human hepatocytes was assessed by measuring intracellular HCV RNA strands. Anti-CD81 antibodies and siRNA-CD81 silencing markedly inhibited (>90%) HCVser infection irrespective of HCV genotype, viral load, or liver donor, while hCD81-large intracellular loop (LEL) had no effect. However, JFH1/HCVcc infection of hepatocytes was modestly inhibited (40 to 60%) by both hCD81-LEL and anti-CD81 antibodies. In conclusion, CD81 is involved in HCVser infection of human hepatocytes, and comparative studies of HCVser versus JFH1/HCVcc infection of human hepatocytes and Huh-7.5 cells revealed that the cell-virion combination is determinant of the entry process.

Dehydroepiandrosterone (DHEA), the major precursor of androgens and estrogens, has several beneficial effects on the immune system, on memory function, and in modulating the effects of diabetes, obesity, and chemical carcinogenesis. Treatment of rats with DHEA influences expression of cytochrome P450 (P450) genes, including peroxisome proliferator-activated receptor alpha (PPAR alpha)- and pregnane X receptor (PXR)-mediated induction of CYP4As and CYP3A23, and suppression of CYP2C11. DHEA treatment elevated the expression and activities of CYP3A4, CYP2C9, CYP2C19, and CYP2B6 in primary cultures of human hepatocytes. Induction of CYP3A4 in human hepatocytes was consistent with studies in rats, but induction of CYP2Cs was unexpected. The role of PXR in this response was studied in transient transfection assays. DHEA activated hPXR in a concentration-dependent manner. Because CYP2B6 induction by DHEA in human hepatocytes might involve either PXR or constitutive androstane receptor (CAR) activation, we performed experiments in primary hepatocytes from CAR knockout mice and observed that CAR was required for maximal induction of Cyp2b10 by DHEA. Furthermore, CAR-mediated Cyp2b10 induction by DHEA was inhibited by the inverse agonist of CAR, androstanol (5 alpha-androstan-3 alpha-ol). Further evidence for CAR activation was provided by cytoplasmic/nuclear transfer of CAR upon DHEA treatment. Elucidation of CAR activation and subsequent induction of CYP2B6 by DHEA presented an additional mechanism by which the sterol can modify the expression of P450s. The effect of DHEA on the activation of the xenosensors PPAR alpha, PXR, and CAR, and the consequent potential for adverse drug/toxicant interactions should be considered in humans treated with this nutriceutical agent.

International audience; Diallyl sulfide (DAS) and diallyl disulfide (DADS) are natural components that could account for the anticarcinogenic properties of garlic, at least in part, through the activation of xenobiotic detoxifying metabolism. The aim of this work was to describe the effect of DAS and DADS on xenobiotic-related gene expressions and to study molecular mechanisms relaying DAS effect. We describe the different effects of DAS and DADS on hepatic CYP2B1/2, CYP3A and epoxide hydrolase (EpH) mRNAs in rats, in terms of activation profile, doses and kinetics. The activation profile varied with the mode of chemical administration, i.e. gastric infusion or intraperitoneal (i.p.) injection. Using gastric infusion, DAS and DADS proved different efficiencies at enhancing the mRNA level of the three drug-metabolizing enzymes. After an i.p. administration, we observed a specific activation of CYP2B1/2 gene by DAS. The DAS-mediated CYP2B1/2 activation occurred at transcriptional level and through an okadaic acid-sensitive pathway. In rat livers, a short sequence (NR1) derived from the CYP2B1/2 promoter was stimulated by DAS and we observed a nuclear accumulation of a DNA-protein complex binding NR1. Because constitutively activated receptor (CAR) is a major transcription factor driving the xenobiotic-induced stimulation of CYP2B1/2 through NR1, the role of CAR as a preferential mediator of DAS effect is discussed.