Nouvelle approche anti-rétrovirale : altération du génome du virus CAS-BR-E MULV à l'intérieur de la cellule hôte

Les rétrovirus humains tels que HTLV-I (Human T-cell Leukemia Virus) et HIV-l (Human lmmunodefiency Virus Type-l) sont associés à des pathologies mortelles et sont largement répandus à travers le monde. Actuellement, aucun vaccin ni thérapie ne sont disponibles pour prévenir ou enrayer totalement l'infection par ces virus. Le but du présent projet est de développer une nouvelle approche anti-rétrovirale se basant sur l'altération de l'ADN viral intégré dans le génome de la cellule hôte. Nous avons utilisé comme modèle le rétrovirus de la leucémie murine (MuLV) Cas-Br-E. Ce virus induit des leucémies non B et non T et engendre une maladie neurologique spongiforme chez certaines souches de souris. La cible du traitement est l'intégrase (IN), une enzyme clé du cycle réplicatif. Elle catalyse l'intégration de l'ADN du virus dans le génome de la cellule de l'hôte et est également impliquée dans la transcription inverse, le transport de l'ADN viral dans le noyau et l'assemblage des particules virales. Les traitements, des ODN mutagènes de l'lN, sont conçus de manière à introduire une mutation au début de la séquence codante de l'IN, engendrant ainsi la production de particules virales défectives et non infectieuses. Les expérimentations se sont effectuées en deux volets: avec le traitement de cellules NIH NE-8 chroniquement infectées par Cas-Br-E et, ensuite, avec celui de cellules NIH 3T3, infectées par Cas-Br-E et traitées à différentes périodes post-infection. L'impact des traitements a été déterminé par titration des virus produits en immunofluorescence, dosage de la transcriptase inverse et séquençage des produits de PCR des ADN et ARN des cellules traitées. Les résultats obtenus avec les ODN mutagènes montrent une diminution de la production de virus infectieux en fonction du nombre de traitements, de la dose utilisée et du moment où les traitements ont été effectués. Une inhibition totale de la production virale est possible avec l'utilisation d'une seule dose massive de 4,0 µM ou d'une faible dose de 1,34 µM ciblée en début d'infection, soit dans les 4 premiers jours. Des mutations du génome ont\ud été observées au niveau de l'ADN et de l'ARN des cellules traitées. En conclusion, la diminution de la production de virus infectieux, suite aux traitements, permet d'envisager l'utilisation de cette stratégie comme nouveIle thérapie contre les infections par HlV-I et HTLV-I, d'autant plus qu'une fois la production de virus infectieux enrayée totalement, l'inhibition se maintient dans le temps

Implication de MEK1 et MEK2 dans l'initiation et la progression du cancer colorectal

Une dérégulation de la voie de signalisation Ras/Raf/MEK/ERK1/2 est observée dans plus de 30% des cancers et des mutations activatrices de RAS sont observées dans 30% à 50% des adénomes colorectaux. À la suite d’une analyse extensive de biopsies de tumeurs colorectales humaines par micromatrices tissulaires (TMA), nous avons observé que 44% des tissus cancéreux exprimaient MEK1/2 phosphorylés, contre 10% des tissus normaux. L'analyse des TMA a également révélé que 79% des tumeurs arboraient un marquage nucléaire de MEK1/2 phosphorylés, contre 4 % pour les tissus normaux. Bien que la voie MEK/ERK1/2 soit fréquemment activée dans les cancers, le rôle précis des isoformes de MEK1 et de MEK2 n'a jamais été clairement établie. De même, l'impact de cette localisation nucléaire aberrante de phospho-MEK1/2, dans l'initiation et la progression des cancers colorectaux, est inconnu. Lors d'un premier projet, nous avons démontré, que l’expression de MEK1 ou MEK2 activé est suffisante pour transformer in vitro des cellules intestinales épithéliales de rat (IEC-6). L'expression des mutants actifs de MEK1 ou MEK2 est suffisante pour induire une dérégulation de la prolifération cellulaire et engendrer la formation d'adénocarcinomes invasifs dans un modèle de greffe orthotopique du côlon chez la souris. Nous avons également démontré que l'inhibition de MEK2 par shRNA supprime complètement la prolifération des lignées humaines de cancer du côlon, alors que la suppression de MEK1 a peu d'effet sur la capacité de prolifération. Le deuxième projet, nous a permis d'observer que l'expression d'un mutant nucléaire de MEK1 dans les cellules IEC-6 transforme drastiquement les cellules. Une augmentation de prolifération, une résistance à l'anoikose, un dérèglement du cycle cellulaire, de l'instabilité chromosomique (CIN), de la tétra/aneuploïdie sont observés. La caractérisation des mécanismes responsables de cette localisation aberrante de MEK1/2 phosphorylés, a permis d'identifier la protéine Sef, un régulateur de la localisation cytoplasmique de MEK/ERK1/2. Nous avons démontré que l'expression d'une forme oncogénique de Ras (H-RasV12) inhibe l'expression de Sef, engendrant alors une accumulation nucléaire de MEK1/2 activés. Plus encore, la réexpression de Sef restaure la localisation cytoplasmique de MEK1/2 et renverse les propriétés tumorigéniques ainsi que l'aneuploïdie induite par Ras activé. Un troisième projet, visant la caractérisation des mécanismes associés à la CIN et à l'aneuploïde engendrés par l'activation aberrante de la voie de Ras-ERK1/2, a permis d'observer que l'hyperactivation de ERK1/2 induit des anomalies mitotiques menant à la binucléation. Une localisation erronée et une surexpression de la kinase Aurora A, de même que des protéines de passage du complexe chromosomique (CPC), Aurora B, Survivine et INCENP, sont observées. L'inhibition partielle de l'activation de ERK1/2 par de faible dose de PD184352, un inhibiteur de MEK1/2, est suffisante pour renverser la surexpression de ces régulateurs mitotiques, de même que corriger les anomalies de la mitose et réduire la tétra/aneuploïdie engendrée par Ras oncogénique. Ainsi, nous avons démontré, pour la première fois, que la voie des MAP kinases ERK1/2 est impliquée dans la CIN, la tétraploïdie et l'aneuploïdie. Nos résultats suggèrent que la perte de Sef est un événement oncogénique précoce, qui contribue à la localisation nucléaire aberrante de MEK1/2 qui est observée dans les tumeurs colorectales. Cette localisation anormale de MEK1/2 est associée à l'initiation de la transformation, la progression tumorale et la CIN, via l'activité soutenue de ERK1/2. Ces informations sont capitales et démontrent l’importance de la voie de signalisation Ras/Raf/MEK/ERK1/2 dans le processus de tumorigénèse colorectale.The Ras-dependent Raf/MEK/ERK1/2 signaling pathway is frequently hyperactivated in human cancer as a result of receptor tyrosine kinase overexpression or gain-of-function mutations in RAS or RAF genes. More specificaly, activating mutation in RAS genes are found in ~ 30-50% of colorectal adenomas and phosphorylation of ERK1/2 is frequently observed in human colorectal cancer cells and tumor specimens. In a large TMA analysis, we found that MEK1/MEK2 are aberrantly activated in 44% of human colorectal cancers. In addition, our analysis revealed that 79% of colorectal cancers exhibit aberrant phospho-MEK1/2 staining in the nucleus, as compared to 4% of normal tissue. How dysregulation and mislocalization of MEK1/2 contribute to tumor initiation and progression is not well understood. In order to determine the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer, wild type and constitutively active forms of MEK1 and MEK2 were ectopically expressed by retroviral gene transfer in the normal intestinal epithelial cell line IEC-6. We found that the expression of activated MEK1 or MEK2 is sufficient to morphologically transform intestinal epithelial cells, dysregulate cell proliferation and induce the formation of high-grade adenocarcinomas after orthotopic transplantation in mice. A large proportion of these intestinal tumors metastasize to the liver and lung. Importantly, we show that silencing of MEK2 expression completely suppresses the proliferation of human colon carcinoma cell lines, whereas inactivation of MEK1 has a much weaker effect. In a second project, we have investigated the impact of the nuclear mislocalization of phosphorylated MEK1/2 observed in colorectal tumors. We show that oncogenic activation of Ras is sufficient to induce the nuclear accumulation of phosphorylated MEK1/2 and ERK1/2 in intestinal epithelial cells. To evaluate the biological impact of the mislocalization of MEK1/2, we have forced the localization of MEK1 in the nucleus of epithelial cells. We found that sustained nuclear MEK1 signaling leads to hyperactivation of ERK1/2 and to enhanced cell proliferation. Nuclear localization of MEK1 also leads to tetraploidization, chromosomal instability (CIN) and tumorigenesis. Importantly, we show that oncogenic Ras downregulates the spatial regulator Sef, concomitant to nuclear accumulation of activated MEK1/2. Moreover, re-expression of Sef is sufficient to restore the normal localization of MEK1/2 and to revert the cell cycle defects and tumorigenesis induced by oncogenic Ras. Another project was initiated to characterize the tetraploidy and CIN observed upon hyperactivation of the Ras-ERK1/2 pathway. Aneuploidy and CIN are observed in the majority of colorectal cancers and are associated with a poorer prognosis. We show that hyperactivation of ERK1/2 by oncogenic Ras or sustained nuclear MEK-ERK1/2 signaling induces mitotic defects that lead to tetraploidy, aneuploidy and CIN. We also found that dysregulation of Ras-ERK1/2 signaling alters the expression and localization of Aurora A and the Chromosomal passenger complex proteins. In conclusion, we show for the first time that the MEK/ERK1/2 signaling pathway is implicated in aneuploidy and CIN. Our results suggest that sustained nuclear ERK1/2 signaling may contribute to the initiation and progression of colorectal cancer by rapidly inducing aneuploidy and CIN. We suggest that loss of Sef is an early oncogenic event that contributes to genetic instability and tumor progression by sustaining nuclear ERK1/2 signaling. These observations are significant and highlight the importance of the Ras-ERK1/2 signaling pathway in colorectal tumorigenesis

Tumour biology of colorectal liver metastasis is a more important factor in survival than surgical margin clearance in the era of modern chemotherapy regimens

AbstractBackgroundThe aim of the authors was to reassess the impact of a positive surgical margin (R1) after a liver resection for colorectal liver metastases (CLMs) on survival in the era of modern chemotherapy, through their own experience and a literature review.MethodsInclusion criteria were: R1 or R0 resection with no local treatment modalities, extra‐hepatic metastases or other cancer.ResultsAmong 337 patients operated between 2000 and 2010, 273 patients were eligible (214 R0/59 R1). The mean follow‐up was 43 ± 29 months. Compared with a R0 resection, a R1 resection offered a lower 5‐year overall (39.1% versus 54.2%, P = 0.010), disease‐free (15.2% versus 31.1%, P = 0.021) and progression‐free (i.e. time to the first non‐curable recurrence; 33.1% versus 47.3%, P = 0.033) survival rates. Metastases in the R1 group were more numerous, larger and more frequently synchronous. Independent factors of poor survival were: number, size and short‐time interval of CLM occurrence, N status, rectal primary, absence of adjuvant chemotherapy, but not a R1 resection. With the more‐systematic administration of chemotherapy since 2005, the intergroup difference in progression‐free survival disappeared (P = 0.264).ConclusionA R1 resection had no prognostic value per se but reflected a more severe disease. The recent change in the prognostic value of a R1 resection may be linked to the beneficial effect of chemotherapy

Violences sexuelles en milieu universitaire au Québec : rapport de recherche de l’enquête ESSIMU

Cette recherche, intitulée « Enquête Sexualité, Sécurité et Interactions en Milieu Universitaire(ESSIMU) : Ce qu’en disent étudiant.es, enseignant.es et employé.es », a permis d’établir un portrait des violences sexuelles se déroulant en contexte universitaire au Québec. Cette enquête d’envergure provinciale regroupe 12 chercheures provenant des 6 universités suivantes : Université du Québec à Montréal, Université de Montréal, Université Laval, Université de Sherbrooke, Université du Québec en Outaouais et Université du Québec à Chicoutimi. L’équipe interdisciplinaire et interuniversitaire compte également le Regroupement québécois des centres d’aide et de lutte contre les agressions à caractère sexuel et le Service aux collectivités de l’UQAM. Afin de documenter les situations de violence sexuelle vécues en milieu universitaire (VSMU), l’équipe ESSIMU a réalisé en 2016 une vaste étude auprès de 9 284 répondant.es dans 6 universités québécoises francophones. Le questionnaire, administré en ligne, a permis de rendre compte d’un large éventail de manifestations de violence sexuelle vécues par l’ensemble de la communauté universitaire (étudiant ou travaillant à l’université). La mesure de victimisation distinguait le harcèlement sexuel (comportements verbaux et non verbaux qui traduisent des attitudes insultantes, hostiles et dégradantes), les comportements sexuels non désirés (comportements verbaux et non verbaux à caractère sexuel, offensants, non désirés ou non réciproques, incluant la tentative de viol et l’agression sexuelle) et la coercition sexuelle (chantage en retour de récompenses)

Activation of MEK1 or MEK2 isoform is sufficient to fully transform intestinal epithelial cells and induce the formation of metastatic tumors

<p>Abstract</p> <p>Background</p> <p>The Ras-dependent ERK1/2 MAP kinase signaling pathway plays a central role in cell proliferation control and is frequently activated in human colorectal cancer. Small-molecule inhibitors of MEK1/MEK2 are therefore viewed as attractive drug candidates for the targeted therapy of this malignancy. However, the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer remains to be established.</p> <p>Methods</p> <p>Wild type and constitutively active forms of MEK1 and MEK2 were ectopically expressed by retroviral gene transfer in the normal intestinal epithelial cell line IEC-6. We studied the impact of MEK1 and MEK2 activation on cellular morphology, cell proliferation, survival, migration, invasiveness, and tumorigenesis in mice. RNA interference was used to test the requirement for MEK1 and MEK2 function in maintaining the proliferation of human colorectal cancer cells.</p> <p>Results</p> <p>We found that expression of activated MEK1 or MEK2 is sufficient to morphologically transform intestinal epithelial cells, dysregulate cell proliferation and induce the formation of high-grade adenocarcinomas after orthotopic transplantation in mice. A large proportion of these intestinal tumors metastasize to the liver and lung. Mechanistically, activation of MEK1 or MEK2 up-regulates the expression of matrix metalloproteinases, promotes invasiveness and protects cells from undergoing anoikis. Importantly, we show that silencing of MEK2 expression completely suppresses the proliferation of human colon carcinoma cell lines, whereas inactivation of MEK1 has a much weaker effect.</p> <p>Conclusion</p> <p>MEK1 and MEK2 isoforms have similar transforming properties and are able to induce the formation of metastatic intestinal tumors in mice. Our results suggest that MEK2 plays a more important role than MEK1 in sustaining the proliferation of human colorectal cancer cells.</p

Early liver transplantation for severe alcohol-related hepatitis not responding to medical treatment: a prospective controlled study

peer reviewedBackground: Early liver transplantation for severe alcohol-related hepatitis is an emerging treatment option. We aimed to assess the risk of alcohol relapse 2 years after early liver transplantation for alcohol-related hepatitis compared with liver transplantation for alcohol-related cirrhosis after at least 6 months of abstinence. Methods: We conducted a multicentre, non-randomised, non-inferiority, controlled study in 19 French and Belgian hospitals. All participants were aged 18 years or older. There were three groups of patients recruited prospectively: patients with severe alcohol-related hepatitis who did not respond to medical treatment and were eligible for early liver transplantation according to a new selection scoring system based on social and addiction items that can be quantified in points (early transplantation group); patients with alcohol-related cirrhosis listed for liver transplantation after at least 6 months of abstinence (standard transplantation group); patients with severe alcohol-related hepatitis not responding to medical treatment not eligible for early liver transplantation according to the selection score (not eligible for early transplantation group), this group did not enter any further liver transplantation processes. We also defined a historical control group of patients with severe alcohol-related hepatitis unresponsive to medical therapy and non-transplanted. The primary outcome was the non-inferiority of 2-year rate of alcohol relapse after transplantation in the early transplantation group compared with the standard transplantation group using the alcohol timeline follow back (TLFB) method and a prespecified non-inferiority margin of 10%. Secondary outcomes were the pattern of alcohol relapse, 2-year survival rate post-transplant in the early transplantation group compared with the standard transplantation group, and 2-year overall survival in the early transplantation group compared with patients in the not eligible for early transplantation group and historical controls. This trial is registered with ClinicalTrials.gov, NCT01756794. Findings: Between Dec 5, 2012, and June 30, 2016, we included 149 patients with severe alcohol-related hepatitis: 102 in the early transplantation group and 47 in the not eligible for early transplantation group. 129 patients were included in the standard transplantation group. 68 patients in the early transplantation group and 93 patients in the standard transplantation group received a liver transplant. 23 (34%) patients relapsed in the early transplantation group, and 23 (25%) patients relapsed in the standard transplantation group; therefore, the non-inferiority of early transplantation versus standard transplantation was not demonstrated (absolute difference 9·1% [95% CI –∞ to 21·1]; p=0·45). The 2-year rate of high alcohol intake was greater in the early transplantation group than the standard transplantation group (absolute difference 16·7% [95% CI 5·8–27·6]) The time spent drinking alcohol was not different between the two groups (standardised difference 0·24 [95% CI −0·07 to 0·55]), but the time spent drinking a large quantity of alcohol was higher in the early transplantation group than the standard transplantation group (standardised difference 0·50 [95% CI 0·17–0·82]). 2-year post-transplant survival was similar between the early transplantation group and the standard transplantation group (hazard ratio [HR] 0·87 [95% CI 0·33–2·26]); 2-year overall survival was higher in the early transplantation group than the not eligible for early transplantation group and historical controls (HR 0·27 [95% CI 0·16–0·47] and 0·21 [0·13–0·32]). Interpretation: We cannot conclude non-inferiority in terms of rate of alcohol relapse post-transplant between early liver transplantation and standard transplantation. High alcohol intake is more frequent after early liver transplantation. This prospective controlled study confirms the important survival benefit related to early liver transplantation for severe alcohol-related hepatitis; and this study provides objective data on survival and alcohol relapse to tailor the management of patients with severe alcohol-related hepatitis. Funding: The present study has been granted by the French Ministry of Health—Programme Hospitalier de Recherche Clinique 2010

ARF6 controls RHOB targeting to endosomes regulating cancer cell invasion

Endocytic trafficking has emerged as an essential mechanism to spatiotemporally coordinate signaling protein complexes that control cytoskeletal dynamics and cell motility. Our study established an unexpected regulatory mechanism whereby ADP ribosylation factors 6 (ARF6) controls the stability and endosomal localization of RAS homologous protein B (RHOB) to regulate cell invasion downstream of the oncogenic receptor tyrosine kinase, MET

Correction: Gresseau et al. A Signaling Crosstalk Links SNAIL to the 37/67 kDa Laminin-1 Receptor Ribosomal Protein SA and Regulates the Acquisition of a Cancer Stem Cell Molecular Signature in U87 Glioblastoma Neurospheres. <i>Cancers</i> 2022, <i>14</i>, 5944

In the original publication [...

A Signaling Crosstalk Links SNAIL to the 37/67 kDa Laminin-1 Receptor Ribosomal Protein SA and Regulates the Acquisition of a Cancer Stem Cell Molecular Signature in U87 Glioblastoma Neurospheres

Background: Three-dimensional in vitro neurospheres cultures recapitulate stemness features associated with poor clinical outcome in glioblastoma patients. They are commonly used to address brain cancer stem cell (CSC) signal transducing biology that regulates spheroids formation and stemness phenotype, and to assess the in vitro pharmacological impact of chemotherapeutic drugs. Objective: Here, we addressed the role of a new signaling axis involved in the regulation of in vitro spheroids formation and assessed the chemopreventive ability of diet-derived epigallocatechin gallate (EGCG) to impact the processes that govern the acquisition of spheroids CSC stemness traits. Methods: Neurospheres were generated from adherent human U87 glioblastoma cancer cell cultures under conditions that recapitulate stemness features. Total RNA and protein lysates were isolated for gene expression by RT-qPCR and protein expression by immunoblot. Transcriptomic analysis was performed through RNA-Seq. Results: Compared to their parental adherent cells, tumorspheres expressed increased levels of the CSC markers NANOG, SOX2, PROM1 (CD133), as well as of the epithelial-to-mesenchymal transition (EMT) markers Fibronectin, SNAI1, and 37/67 kDa laminin-1 receptor ribosomal protein SA (RPSA). Increased PROM1, SOX2, Fibronectin, and RPSA transcripts level were also observed in clinical grade IV glioblastoma tissues compared to normal tissue. EGCG treatment reduced dose-dependently tumorspheres size and inhibited the transcriptional regulation of those genes. An apoptotic signature was also found in spheroids with increased signal transducing events involving GSK3&alpha;/&beta;, RSK, and CREB. These were repressed upon RPSA gene silencing and partially by SNAI1 silencing. Conclusion: This work highlights a signaling axis linking RPSA upstream of SNAIL in neurospheres genesis and supports the chemopreventive impact that diet-derived EGCG may exert on the acquisition of CSC traits

Evaluation des haies nouvelles plantées par l’association Terres et Bocages

Evaluation des haies nouvelles plantées par l’association Terres et Bocages. Journée Terres et Bocag