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Endoplasmic Reticulum (ER) stress is a hallmark of various diseases. Cells cope with ER stress through the activation of an adaptive signaling pathway named the Unfolded Protein Response (UPR) which is mediated by three ER-resident sensors. The most evolutionary conserved of the UPR sensors is IRE1alpha that elicits diverse downstream effects through its kinase and endoribonuclease (RNase) activities. IRE1alpha RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through Regulated IRE1-Dependent Decay (RIDD). The degree of exertion of these two activities plays an instrumental role in cells life and death decisions upon ER stress. Until now, the biochemical and biological outputs of IRE1alpha RNase activity have been well documented, however, the precise mechanisms controlling whether IRE1 signaling is adaptive or pro-death (terminal) remain unclear. This prompted us to further investigate those mechanisms and we hypothesized that XBP1 mRNA splicing and RIDD activity could be co-regulated within the context of the IRE1alpha RNase regulatory network. We showed that a key nexus in this pathway is the tRNA ligase RtcB which, together with IRE1alpha is responsible for XBP1 mRNA splicing. We demonstrated that RtcB is tyrosine phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we identified RtcB Y306 as a key residue which, when phosphorylated, perturbs RtcB interaction with IRE1alpha, thereby attenuating XBP1 mRNA splicing and favoring RIDD. Our results demonstrate that the IRE1alpha RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and depending on the nature of the stress determines cell adaptive or death outputs.

International audience; Proteostasis imbalance is emerging as a major hallmark of cancer, driving tumor aggressiveness. Evidence suggests that the endoplasmic reticulum (ER), a major site for protein folding and quality control, plays a critical role in cancer development. This concept is valid in glioblastoma multiform (GBM), the most lethal primary brain cancer with no effective treatment. We previously demonstrated that the ER stress sensor IRE1 alpha (referred to as IRE1) contributes to GBM progression, through XBP1 mRNA splicing and regulated IRE1-dependent decay (RIDD) of RNA. Here, we first demonstrated IRE1 signaling significance to human GBM and defined specific IRE1-dependent gene expression signatures that were confronted to human GBM transcriptomes. This approach allowed us to demonstrate the antagonistic roles of XBP1 mRNA splicing and RIDD on tumor outcomes, mainly through selective remodeling of the tumor stroma. This study provides the first demonstration of a dual role of IRE1 downstream signaling in cancer and opens a new therapeutic window to abrogate tumor progression.

MicroRNAs (miRNA) are generally described as negative regulators of gene expression. However, some evidence suggests that they may also play positive roles. As such, we reported that miR-1291 leads to a GPC3 mRNA expression increase in hepatoma cells through a 3′ untranslated region (UTR)-dependent mechanism. In the absence of any direct interaction between miR-1291 and GPC3 mRNA, we hypothesized that miR-1291 could act by silencing a negative regulator of GPC3 mRNA expression. Based on in silico predictions and experimental validation, we demonstrate herein that miR-1291 represses the expression of the mRNA encoding the endoplasmic reticulum (ER)-resident stress sensor IRE1α by interacting with a specific site located in the 5′ UTR. Moreover, we show, in vitro and in cultured cells, that IRE1α cleaves GPC3 mRNA at a 3′ UTR consensus site independently of ER stress, thereby prompting GPC3 mRNA degradation. Finally, we show that the expression of a miR-1291-resistant form of IRE1α abrogates the positive effects of miR-1291 on GPC3 mRNA expression. Collectively, our data demonstrate that miR-1291 is a biologically relevant regulator of GPC3 expression in hepatoma cells and acts through silencing of the ER stress sensor IRE1α.

International audience; Hepatocellular carcinoma (HCC) is the major primary liver cancer. Glypican-3 (GPC3), one of the most abnormally expressed genes in HCC, participates in liver carcinogenesis. Based on data showing that GPC3 expression is posttranscriptionally altered in HCC cells compared to primary hepatocytes, we investigated the implication of microRNAs (miRNAs) in GPC3 overexpression and HCC. To identify GPC3-regulating miRNAs, we developed a dual-fluorescence FunREG (functional, integrated, and quantitative method to measure posttranscriptional regulations) system that allowed us to screen a library of 876 individual miRNAs. Expression of candidate miRNAs and that of GPC3 messenger RNA (mRNA) was measured in 21 nontumoral liver and 112 HCC samples. We then characterized the phenotypic consequences of modulating expression of one candidate miRNA in HuH7 cells and deciphered the molecular mechanism by which this miRNA controls the posttranscriptional regulation of GPC3. We identified five miRNAs targeting GPC3 3'-untranslated region (UTR) and regulating its expression about the 876 tested. Whereas miR-96 and its paralog miR-1271 repressed GPC3 expression, miR-129-1-3p, miR-1291, and miR-1303 had an inducible effect. We report that miR-1271 expression is down-regulated in HCC tumor samples and inversely correlates with GPC3 mRNA expression in a particular subgroup of HCC. We also report that miR-1271 inhibits the growth of HCC cells in a GPC3-dependent manner and induces cell death.CONCLUSION:Using a functional screen, we found that miR-96, miR-129-1-3p, miR-1271, miR-1291, and miR-1303 differentially control GPC3 expression in HCC cells. In a subgroup of HCC, the up-regulation of GPC3 was associated with a concomitant down-regulation of its repressor miR-1271. Therefore, we propose that GPC3 overexpression and its associated oncogenic effects are linked to the down-regulation of miR-1271 in HCC.

Introduction:Glioblastoma multiforme (GBM) is the most lethal form of glioma with an overall survival at 5 years nearly null (< 5%). Increasing evidences point towards the RNase activity of IRE1 as a central player in GBM development, particularly in cancer cell invasion, tumor vascularization and recruitment of inflammatory or immune cells. Indeed, IRE1 RNase promotes XBP1 mRNA splicing, a well-described cytoprotective transcription factor that is required for cancer cell survival and this activity also contributes to the degradation of mRNA and microRNA, an activity called RIDD. Recent studies unraveled the presence of somatic mutations on the IRE1 gene in GBM that could play a driver role but without providing any functional information.Results:Here, we have sequenced IRE1 exons in 23 human GBMs and identified a new somatic mutation on the IRE1 gene. Interestingly, using biochemical approaches and an orthotopic tumor graft mouse model we show that this variant displays an altered IRE1 RNase activity. IRE1-dependent gene expression pattern alterations lead to major functional consequences in terms of tumor phenotype including (i) a magnified induction of the inflammatory and pro-angiogenic pathways based on transcriptome signatures from human tumors, (ii) an increase of the aggressiveness/infiltration potential of U87 derived tumors in mice and (iii) the modulation of immune cells recruitment to the tumor.Conclusions:This study provides the first evidence that the selectivity of IRE1 RNase can be modulated naturally and unravels the first mechanistic example of how a somatic mutation in the IRE gene can provide adaptive advantages to glioblastoma cells by reprogramming the tumor stroma.

International audience; Besides the fact that miR-96 and miR-182 belong to the miR-182/183 cluster, their seed region (UUGGCA, nucleotides 2-7) is identical suggesting potential common properties in mRNA target recognition and cellular functions. Here, we used the mRNA encoding Glypican-3, a heparan-sulfate proteoglycan, as a model target as its short 3' untranslated region is predicted to contain one miR-96/182 site, and assessed whether it is post-transcriptionally regulated by these two microRNAs. We found that miR-96 downregulated GPC3 expression by targeting its mRNA 3'-untranslated region and interacting with the predicted site. This downregulatory effect was due to an increased mRNA degradation and depended on Argonaute-2. Despite its seed similarity with miR-96, miR-182 was unable to regulate GPC3. This differential regulation was confirmed on two other targets, FOXO1 and FN1. By site-directed mutagenesis, we demonstrated that the miRNA nucleotide 8, immediately downstream the UUGGCA seed, plays a critical role in target recognition by miR-96 and miR-182. Our data suggest that because of a base difference at miRNA position 8, these two microRNAs control a completely different set of genes and therefore are functionally independent.

An increasing number of arguments, including altered microRNA expression, support the idea that post-transcriptional deregulation participates in gene disturbances found in diseased tissues. To evaluate this hypothesis, we developed a method which facilitates post-transcriptional investigations in a wide range of human cells and experimental conditions. This method, called FunREG (functional, integrated and quantitative method to measure post-transcriptional regulation), connects lentiviral transduction with a fluorescent reporter system and quantitative PCR. Using FunREG, we efficiently measured post-transcriptional regulation mediated either by selected RNA sequences or regulatory factors (microRNAs), and then evaluated the contribution of mRNA decay and translation efficiency in the observed regulation. We demonstrated the existence of gene-specific post-transcriptional deregulation in liver tumour cells, and also reported a molecular link between a transcript variant abrogating HDAC6 (histone deacetylase 6) regulation by miR-433 and a rare familial genetic disease. Because FunREG is sensitive, quantitative and easy to use, many applications can be envisioned in fundamental and pathophysiological research.

Background The aim of this study was to assess the psychological consequences of HRCT scan screening in retired asbestos-exposed workers. Methods A HRCT-scan screening program for asbestos-related diseases was carried out in four regions of France. At baseline (T1), subjects filled in self-administered occupational questionnaires. In two of the regions, subjects also received a validated psychological scale, namely the psychological consequences questionnaire (PCQ). The physician was required to provide the subject with the results of the HRCT scan at a final visit. A second assessment of psychological consequences was performed 6 months after the HRCT-scan examination (T2). PCQ scores were compared quantitatively (t-test, general linear model) and qualitatively (chi²-test, logistic regression) to screening results. Multivariate analyses were adjusted for gender, age, smoking, asbestos exposure and counseling. Results Among the 832 subjects included in this psychological impact study, HRCT-scan screening was associated with a significant increase of the psychological score 6 months after the examination relative to baseline values (8.31 to 10.08, p < 0.0001, t-test). This increase concerned patients with an abnormal HRCT-scan result, regardless of the abnormalities, but also patients with normal HRCT-scans after adjustment for age, gender, smoking status, asbestos exposure and counseling visit. The greatest increase was observed for pleural plaques (+3.60; 95%CI [+2.15;+5.06]), which are benign lesions. Detection of isolated pulmonary nodules was also associated with a less marked but nevertheless significant increase of distress (+1.88; 95%CI [+0.34;+3.42]). However, analyses based on logistic regressions only showed a close to significant increase of the proportion of subjects with abnormal PCQ scores at T2 for patients with asbestosis (OR = 1.92; 95%CI [0.97-3.81]) or with two or more diseases (OR = 2.04; 95%CI [0.95-4.37]). Conclusion This study suggests that HRCT-scan screening may be associated with increased distress in asbestos-exposed subjects. If confirmed, these results may have consequences for HRCT-scan screening recommendations.