miR‐4510 blocks hepatocellular carcinoma development through RAF1 targeting and RAS/RAF/MEK/ERK signalling inactivation

International audienceBACKGROUND:Therapeutic outcomes using the multikinase inhibitors, sorafenib and regorafenib, remain unsatisfactory for patients with advanced hepatocellular carcinoma (HCC). Thus, new drug modalities are needed. We recently reported the remarkable capacity of miR-4510 to impede the growth of HCC and hepatoblastoma through Glypican-3 (GPC3) targeting and Wnt pathway inactivation.METHODS:To identify new targets of miR-4510, we used a label-free proteomic approach and reported down-regulation of RAF proto-oncogene serine/threonine-protein kinase (RAF1) by miR-4510. Because the tumourigenic role of RAF1 in HCC is controversial, we further studied RAF1:miR-4510 interactions using cellular, molecular as well as functional approaches and a chicken chorioallantoic membrane (CAM) xenograft model.RESULTS:We found an increase in RAF1 protein in 59.3% of HCC patients and a specific up-regulation of its transcript in proliferative tumours. We showed that miR-4510 inactivates the RAS/RAF/MEK/ERK pathway and reduces the expression of downstream targets (ie c-Fos proto-oncogene [FOS]) through RAF1 direct targeting. At a cellular level, miR-4510 inhibited HCC cell proliferation and migration and induced senescence in part by lowering RAF1 messenger RNA (mRNA) and protein expression. Finally, we confirmed the pro-tumoural function of RAF1 protein in HCC cells and its ability to sustain HCC tumour progression in vitro and in vivo.CONCLUSIONS:In this work, we confirm that RAF1 acts as an oncogene in HCC and further demonstrate that miR-4510 acts as a strong tumour suppressor in the liver by targeting many proto-oncogenes, including GPC3 and RAF1, and subsequently controlling key biological and signalling pathways among which Wnt and RAS/RAF/MEK/ERK signals

Proliferation Genes Repressed by TGF-β AreDownstream of Slug/Snail2 in Normal Bronchial EpithelialProgenitors and Are Deregulated in COPD

International audienceSlug/Snail2 belongs to the Epithelial-Mesenchymal Transition (EMT)-inducing transcription factors involved in development and diseases. Slug is expressed in adult stem/progenitor cells of several epithelia, making it unique among these transcription factors. To investigate Slug role in human bronchial epithelium progenitors, we studied primary bronchial basal/progenitor cells in an air-liquid interface culture system that allows regenerating a bronchial epithelium. To identify Slug downstream genes we knocked down Slug in basal/progenitor cells from normal subjects and subjects with COPD, a respiratory disease presenting anomalies in the bronchial epithelium and high levels of TGF-β in the lungs. We show that normal and COPD bronchial basal/progenitors, even when treated with TGF-β, express both epithelial and mesenchymal markers, and that the epithelial marker E-cadherin is not a target of Slug and, moreover, positively correlates with Slug. We reveal that Slug downstream genes responding to both differentiation and TGF-β are different in normal and COPD progenitors, with in particular a set of proliferation-related genes that are among the genes repressed downstream of Slug in normal but not COPD. In COPD progenitors at the onset of differentiation in presence of TGF-β, we show that there is positive correlations between the effect of differentiation and TGF-β on proliferation-related genes and on Slug protein, and that their expression levels are higher than in normal cells. As well, the expression of Smad3 and β-Catenin, two molecules from TGF-β signaling pathways, are higher in COPD progenitors, and our results indicate that proliferation-related genes and Slug protein are increased by different TGF-β-induced mechanisms

Proliferation Genes Repressed by TGF-β AreDownstream of Slug/Snail2 in Normal Bronchial EpithelialProgenitors and Are Deregulated in COPD

International audienceSlug/Snail2 belongs to the Epithelial-Mesenchymal Transition (EMT)-inducing transcription factors involved in development and diseases. Slug is expressed in adult stem/progenitor cells of several epithelia, making it unique among these transcription factors. To investigate Slug role in human bronchial epithelium progenitors, we studied primary bronchial basal/progenitor cells in an air-liquid interface culture system that allows regenerating a bronchial epithelium. To identify Slug downstream genes we knocked down Slug in basal/progenitor cells from normal subjects and subjects with COPD, a respiratory disease presenting anomalies in the bronchial epithelium and high levels of TGF-β in the lungs. We show that normal and COPD bronchial basal/progenitors, even when treated with TGF-β, express both epithelial and mesenchymal markers, and that the epithelial marker E-cadherin is not a target of Slug and, moreover, positively correlates with Slug. We reveal that Slug downstream genes responding to both differentiation and TGF-β are different in normal and COPD progenitors, with in particular a set of proliferation-related genes that are among the genes repressed downstream of Slug in normal but not COPD. In COPD progenitors at the onset of differentiation in presence of TGF-β, we show that there is positive correlations between the effect of differentiation and TGF-β on proliferation-related genes and on Slug protein, and that their expression levels are higher than in normal cells. As well, the expression of Smad3 and β-Catenin, two molecules from TGF-β signaling pathways, are higher in COPD progenitors, and our results indicate that proliferation-related genes and Slug protein are increased by different TGF-β-induced mechanisms

Validating intestinal effects of food-grade titanium dioxide using a murine gut organoid model as alternative to in vivo models

International audienceBackground: Nanoparticles (NPs) found in the human diet mainly originate from inorganic food additives, often used quantum satis in common foodstuff, which raises public health concerns due to daily exposure. The whitener and opacifying agent titanium dioxide (TiO2, E171 in EU) is one of the most studied nanomaterial, evoking inflammatory responses and precancerous lesions in the rodent intestine. Investigating the potential hazards of chronic oral exposure to NPs is often time-consuming and requires animal models, specific spaces and skills. However, recent technical advances in stem cells and threedimensional cultures allowed the use of organoids as an alternative model to in vivo experiments. Herein we used murine intestinal organoids to characterize intestinal impacts of food-grade TiO2 in comparison to already reported in vivo data, and to validate organoids as a reliable model for studying the effects of foodborne NPs in the gut.Methods: Three different wild-type C57bl/6 mice were used for small intestine collection. Intestinal crypts were purified, dissociated, and cells were cultured for organoid growth. After 4 passages, organoids were dissociated and seeded as a 2.5D culture, then exposed to 0.1, 1, 10 or 100µg/ml of E171 for 24h. Supernatants were collected, and cytotoxicity assessed by LDH release quantification. Total RNA was extracted from samples and analyzed for cell proliferation and differentiation, genotoxicity, antimicrobial peptides, permeability, oxidative stress, Toll Like Receptors (TLR), NFκB, cytokine and chemokine gene expressions by qPCR. Cell apoptosis was also evaluated by cleaved Caspase-3 quantification using immunofluorescence.Results: Gut organoids exposed to E171 showed a dose-dependent up-regulation of the cell proliferation marker Mki67 together with increased protein expression of cleaved-Caspase-3, suggesting epithelium renewal or restructuring. This occurred in parallel to a decreased expression of the enterocyte differentiation markers Alpi and Krt20 as well as up-regulation of the neuroendocrine marker Chga. Moreover, food-grade E171 decreased gene expression of antimicrobial peptides (Lyz, Reg3b, S100a8) and tight junction proteins (F11r, Tjp1, Ocln, Cldn7, Cldn15), suggesting altered epithelial secretion and permeability. We also showed that the TLR4-NFκB pathway was negatively impacted in a dose-dependent manner, while oxidative stress, cytokine and chemokine gene expressions remained unaltered. Although E171 exposure was not cytotoxic, TiO2 increased expression of gadd45a at low dose (i.e. 1µg/ml), suggesting DNA damage.Conclusions: Taking together, a 24h-exposure of murine intestinal organoids to food-grade TiO2 impacts epithelial barrier integrity (cell proliferation and differentiation, gut permeability, genotoxic effect) and antimicrobial defenses as reported in vivo in rodent models, hence validating the use of intestinal organoids for toxicological studies of foodborne NPs

Validating Enteroid-Derived Monolayers from Murine Gut Organoids for Toxicological Testing of Inorganic Particles: Proof-of-Concept with Food-Grade Titanium Dioxide

Human exposure to foodborne inorganic nanoparticles (NPs) is a growing concern. However, identifying potential hazards linked to NP ingestion often requires long-term exposure in animals. Owing these constraints, intestinal organoids are a promising alternative to in vivo experiments; as such, an in vitro approach should enable a rapid and reliable assessment of the effects of ingested chemicals on the gut. However, this remains to be validated for inorganic substances. In our study, a transcriptomic analysis and immunofluorescence staining were performed to compare the effects of food-grade TiO2 (fg-TiO2) on enteroid-derived monolayers (EDMs) from murine intestinal organoids to the known impacts of TiO2 on intestinal epithelium. After their ability to respond to a pro-inflammatory cytokine cocktail was validated, EDMs were exposed to 0, 0.1, 1, or 10 µg fg-TiO2/mL for 24 h. A dose-related increase of the muc2, vilin 1, and chromogranin A gene markers of cell differentiation was observed. In addition, fg-TiO2 induced apoptosis and dose-dependent genotoxicity, while a decreased expression of genes encoding for antimicrobial peptides, and of genes related to tight junction function, was observed. These results validated the use of EDMs as a reliable model for the toxicity testing of foodborne NPs likely to affect the intestinal barrier

Proliferation genes repressed by TGF-β are downstream of Slug/Snail2 in normal bronchial epithelial progenitors and are deregulated in COPD

Abstract Slug/Snail2 belongs to the Epithelial-Mesenchymal Transition (EMT)-inducing transcription factors involved in development and diseases. Slug is expressed in adult stem/progenitor cells of several epithelia, making it unique among these transcription factors. To investigate Slug role in human bronchial epithelium progenitors, we studied primary bronchial basal/progenitor cells in an air-liquid interface culture system that allows regenerating a bronchial epithelium. To identify Slug downstream genes we knocked down Slug in basal/progenitor cells from normal subjects and subjects with COPD, a respiratory disease presenting anomalies in the bronchial epithelium and high levels of TGF-β in the lungs. We show that normal and COPD bronchial basal/progenitors, even when treated with TGF-β, express both epithelial and mesenchymal markers, and that the epithelial marker E-cadherin is not a target of Slug and, moreover, positively correlates with Slug. We reveal that Slug downstream genes responding to both differentiation and TGF-β are different in normal and COPD progenitors, with in particular a set of proliferation-related genes that are among the genes repressed downstream of Slug in normal but not COPD. In COPD progenitors at the onset of differentiation in presence of TGF-β, we show that there is positive correlations between the effect of differentiation and TGF-β on proliferation-related genes and on Slug protein, and that their expression levels are higher than in normal cells. As well, the expression of Smad3 and β-Catenin, two molecules from TGF-β signaling pathways, are higher in COPD progenitors, and our results indicate that proliferation-related genes and Slug protein are increased by different TGF-β-induced mechanisms

Successful efavirenz dose reduction guided by therapeutic drug monitoring

Background: There are potential benefits to individualizing dosage in patients treated with efavirenz (EFV). We tested a simplified algorithm based on a Bayesian pharmacokinetic approach for guiding dose reduction in patients with EFV concentrations above the 75th percentile (P75) with documented virological efficacy. Methods: We designed a prospective, open-label, multicentre study. All consenting participants with EFV concentrations above P75 on standard dosage were included in a dose-reduction cycle. Primary end point was the number of patients who reached plasma concentrations within target (1,000-4,000 ng/ml) after, at most, two cycles of dose reduction at 3 and 6 months. CYP2B6 genetic characterization was performed. Results: Seventy-two patients were screened and 13 fulfilled selection criteria. These patients, with undetectable viraemia on a stable 600 mg EFV-based regimen, had a median (interquartile range) EFV plasma level of 8,112 ng/ml (5,993-10,278) at baseline; 38% (between P75 and P95) qualified for a 400 mg EFV dose, and 62% (above P95) qualified for a 200 mg EFV dose. After one to two dose-reduction cycles, all patients reached targets for EFV plasma concentration at 24 weeks. The predictive dose reduction based on genetic profile differed from dose reduction according to therapeutic drug monitoring (TDM) in three patients. All patients maintained viral suppression at 6 months. Conclusions: A standardized TDM-guided EFV dose-reduction strategy over a 24-week period was successful, safe and yielded EFV plasma concentrations within the recommended therapeutic range. In addition to improving neuropsychiatric tolerability, EFV dose reduction has the potential to substantially decrease treatment cost.</p

Prognostic Risk Classification for Biochemical Relapse-Free Survival in Oligometastatic Recurrent Prostate Cancer Determined by Choline PET

International audienc

A posteriori dietary patterns better explain variations of the gut microbiome than individual markers in the American Gut Project

BackgroundIndividual diet components and specific dietary regimens have been shown to impact the gut microbiome.ObjectivesHere, we explored the contribution of long-term diet by searching for dietary patterns that would best associate with the gut microbiome in a population-based cohort.MethodsUsing a priori and a posteriori approaches, we constructed dietary patterns from an FFQ completed by 1800 adults in the American Gut Project. Dietary patterns were defined as groups of participants or combinations of food variables (factors) driven by criteria ranging from individual nutrients to overall diet. We associated these patterns with 16S ribosomal RNA-based gut microbiome data for a subset of 744 participants.ResultsCompared to individual features (e.g., fiber and protein), or to factors representing a reduced number of dietary features, 5 a posteriori dietary patterns based on food groups were best associated with gut microbiome beta diversity (P&nbsp;≤&nbsp;0.0002). Two patterns followed Prudent-like diets-Plant-Based and Flexitarian-and exhibited the highest Healthy Eating Index 2010 (HEI-2010) scores. Two other patterns presented Western-like diets with a gradient in HEI-2010 scores. A fifth pattern consisted mostly of participants following an Exclusion diet (e.g., low carbohydrate). Notably, gut microbiome alpha diversity was significantly lower in the most Western pattern compared to the Flexitarian pattern (P&nbsp;≤&nbsp;0.009), and the Exclusion diet pattern was associated with low relative abundance of Bifidobacterium (P&nbsp;≤&nbsp;1.2&nbsp;×&nbsp;10-7), which was better explained by diet than health status.ConclusionsWe demonstrated that global-diet a posteriori patterns were more associated with gut microbiome variations than individual dietary features among adults in the United States. These results confirm that evaluating diet as a whole is important when studying the gut microbiome. It will also facilitate the design of more personalized dietary strategies in general populations

Human TR146 cells and pig buccal mucosa to assess oral transmucosal passage and buccal toxicity of foodgrade titanium dioxide

International audienceBackground: Today, the use of titanium dioxide (TiO 2) as food additive (E171) has been banned by the European Commission, due to concerns for human health based on studies showing TiO 2 particles systematically available, tissue accumulation, a genotoxic risk and possible promotion of precancerous lesions. However, E171 is still present in toothpastes and pharmaceutical tablets as a whitening powder mixing nano-and submicronic particles. Risk assessment of TiO 2 intake by oral route is mainly based on the assumption that particles are mainly absorbed by the intestine. However, while the buccal mucosa is the first exposed area, the possibility of an oro-transmucosal passage has not been documented so far. In order to gain insight on possible adverse effects for human health associated to E171 buccal exposure, we analyzed in vivo the translocation of TiO 2 (E171) in the buccal mucosa of pigs used as human mouth model. Moreover, we evaluated in vitro the particle translocation on human buccal TR146 cell line, and measured cytotoxic and genotoxic effects on proliferative and differentiated epithelial cells. Methods & Results: Under realistic exposure conditions with 50 µg/ml of food-grade TiO 2 in water suspension (size distribution 20-440 nm; mean size of 105 nm) deposited under the tongue of pigs, TEM-EDX data revealed the presence of small aggregates of TiO 2 particles translocated into the buccal mucosa from 30 minutes of exposure, reaching submaxillary lymph nodes after 4 hours. In human TR146 cells exposed to E171, kinetic analysis using confocal, TEM and SIMS imaging showed progressive and large uptake of isolated or small aggregates of both submicronic and nanosized particles, showing high permeability capacity. At 2h of E171 exposure, cytotoxicity, genotoxicity and oxidative stress were investigated on both proliferative or differentiated TR146 cells, in comparison with two TiO 2 size standards of 115 nm and 21 nm in diameter. All tested TiO 2 particles were reported cytotoxic on proliferative TR146 cells, and this effect was almost abolished following differentiation. Oxidative stress and genotoxicity assessed through γH2AX and 53BP1 foci formation and comet assay were only reported for E171 sample and TiO 2 particles of 115 nm, suggesting the particles above 20 nm responsive of these effects, and mainly on proliferative cells. Conclusions: Taken together, these results show in vivo and in vitro that the buccal mucosa is an important absorption route for systemic passage of foodborne TiO 2 (E171) particles. In human cells, uptake of TiO2 particles was cytotoxic without size effects, while they generate further oxidative and genotoxic stresses in proliferative buccal cells, that could impair epithelium renewal in the mouth. Altogether, these data emphasize that buccal exposure should be considered for toxicokinetic and risk assessments of TiO2 in human when used as food additive, including in toothpaste and pharmaceutical formulations