CDK8 : une cible de la voie KRAS/MAP Kinase dans la carcinogénèse colorectale

La voie KRAS/BRAF/MEK/ERK MAP Kinase joue un rôle clé dans le contrôle de la prolifération des cellules épithéliales intestinales normales et cancéreuses. En effet, on retrouve des mutations du gène KRAS dans près de 35 à 40% des cancers colorectaux et une mutation du gène BRAF dans 10 à 15% des cas. Ces mutations de type gain-de-fonction sont mutuellement exclusives, ce qui suggère que la signalisation MEK/ERK qui est en aval de BRAF joue possiblement un rôle crucial dans le développement de plus de 60% des cancers colorectaux. Notre laboratoire a d’ailleurs rapporté que l’expression d’une forme mutante hyperactive de MEK1 est suffisante pour induire la transformation des cellules épithéliales intestinales normales en culture. Cette transformation est caractérisée par une transition épithélium-mésenchyme (EMT) conférant aux cellules des capacités tumorales, invasives et métastatiques. Afin de mieux comprendre les mécanismes moléculaires impliqués dans les effets transformant de MEK1, une analyse comparative par micropuces d’ADN (Affymetrix) a été effectuée et celle-ci a montré que le gène codant pour la protéine CDK8, une kinase dépendante des cyclines, est un des gènes les plus induits (12 fois) par l’hyperactivation de MEK1. Ce résultat suggèrerait l’implication de CDK8 dans l’oncogenèse colorectale induite par l’hyperactivation de la voie KRAS/MAP Kinase. De manière intéressante, nous avons d’abord mis en évidence que CDK8 était surexprimée dans des tumeurs de patients atteints de cancer colorectal de différents stades ainsi que dans des lignées cancéreuses colorectales humaines. Parmi ces lignées cellulaires analysées, nous avons mis en évidence que cette surexpression était en partie dépendante de l’activité MEK. Nous avons aussi confirmé la surexpression de CDK8 dans des lignées de cellules épithéliales intestinales de rat exprimant les oncogènes KRAS ou BRAF ou le mutant de MEK1 constitutivement actif. La baisse d’expression de CDK8 par l’utilisation d’un shARN a révélé que CDK8 contribue à l’hyperprolifération cellulaire ainsi qu’à la croissance en indépendance d’ancrage induite par l’expression du mutant hyperactif de MEK1. De plus, la baisse d’expression de CDK8 atténue le phénotype fibroblastique des cellules transformées par l’oncogène BRAF ou le mutant de MEK1 constitutivement actif, qui exhibent un phénotype plus épithélial. Nous avons pu mettre en évidence que CDK8 serait impliqué dans l’expression de gènes liés à la morphologie cellulaire tel que Snail1, Snail2 et Gem. Nos résultats montrent donc que CDK8 contribue au potentiel oncogénique de la voie MAP Kinase dans les cellules épithéliales intestinales en modulant leurs capacités prolifératives et leur transformation morphologique

High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies

Genetic investigations of X-linked intellectual disabilities have implicated the ARX (Aristaless-related homeobox) gene in a wide spectrum of disorders extending from phenotypes characterised by severe neuronal migration defects such as lissencephaly, to mild or moderate forms of mental retardation without apparent brain abnormalities but with associated features of dystonia and epilepsy. Analysis of Arx spatio-temporal localisation profile in mouse revealed expression in telencephalic structures, mainly restricted to populations of GABAergic neurons at all stages of development. Furthermore, studies of the effects of ARX loss of function in humans and animal models revealed varying defects, suggesting multiple roles of this gene during brain development. However, to date, little is known about how ARX functions as a transcription factor and the nature of its targets. To better understand its role, we combined chromatin immunoprecipitation and mRNA expression with microarray analysis and identified a total of 1006 gene promoters bound by Arx in transfected neuroblastoma (N2a) cells and in mouse embryonic brain. Approximately 24% of Arx-bound genes were found to show expression changes following Arx overexpression or knock-down. Several of the Arx target genes we identified are known to be important for a variety of functions in brain development and some of them suggest new functions for Arx. Overall, these results identified multiple new candidate targets for Arx and should help to better understand the pathophysiological mechanisms of intellectual disability and epilepsy associated with ARX mutations

P2Y6 receptor, an actor of colorectal tumorigenesis

Le cancer colorectal (CCR) est le 3e cancer le plus répandu dans le monde. La carcinogenèse colorectale est un processus qui s’établit lentement via une succession d’altérations génétiques. Les maladies inflammatoires intestinales (MII) sont des facteurs de risques pour l’établissement de la carcinogenèse colorectale, puisque 20% des CCR sont liés à une inflammation chronique. Le microenvironnement de ces tumeurs est riche en cellules immunitaires, comme les neutrophiles, en molécules immunomodulatrices (TNFα, CXCL8), ainsi qu’en nucléotides (ATP, UDP) relâchés par les cellules en état de stress, incluant les cellules épithéliales intestinales (CEI). L’UDP est l’agoniste unique du récepteur P2Y6 (P2Y6R). Des études précédentes réalisées par notre équipe ont mis en évidence le rôle de ce récepteur dans l’inflammation intestinale. Dans cette étude nous avons choisis de nous concentrer sur le rôle de P2Y6R dans la carcinogenèse colorectale. Nous avons premièrement, mis en évidence dans un modèle in vivo de souris invalidées pour P2Y6R que l’absence de ce récepteur réduisait le potentiel tumorigénique des CEI dans le modèle AOM-DSS. P2Y6R pourrait jouer un rôle dans la néovascularisation des tumeurs, ainsi que dans la prolifération tumorale, en favorisant une activation de la voie Wnt/β-caténine entraînant l’expression de ces gènes cibles tels que c-Myc. P2Y6R pourrait également jouer un rôle anti-apoptotique et ainsi favoriser la croissance tumorale. L’activation de P2Y6R induit une résistance à l’apoptose induite par le TNFα dans les cellules cancéreuses colorectales, HT-29. Ceci, notamment en favorisant la stabilisation de l’expression de la protéine anti-apoptotique XIAP. Ce rôle de résistance à la mort cellulaire a également été mis en évidence en réponse au 5-FU dans des organoïdes de tumeurs de côlon de souris induites par AOM-DSS. Deuxièmement, nous avons mis en évidence que P2Y6R jouait un rôle dans le maintien de l’intégrité de la barrière épithéliale. P2Y6R serait impliqué dans le processus de cicatrisation, notamment en favorisant la sécrétion de cytokines permettant la migration des CEI. P2Y6R pourrait également jouer un rôle sur la qualité de la couche de mucus protecteur de l’épithélium intestinal. Enfin, nous avons également montré que P2Y6R participait au recrutement des neutrophiles qui sont des cellules impliquées dans l’immunité innée et qui peuvent favoriser la tumorigenèse en participant à la cascade métastatique et en influençant l’angiogenèse. Cette étude met en évidence que P2Y6R pourrait être un oncogène, intervenant dans divers mécanismes pouvant favoriser la carcinogenèse colorectale.Abstract : Colorectal cancer (CRC) is the third most prevalent form of cancer in the world. Colorectal carcinogenesis is a slow process which is established by a succession of genetics alterations. Intestinal bowel diseases (IBD) can promote colorectal carcinogenesis since 20% of CRC is the result of chronic intestinal inflammation. The environment surrounding tumors is rich in immune cells, like neutrophils, in immunomodulatory molecules (TNFα, CXCL8), and nucleotides (ATP, UDP) that are released by stressed cells such as intestinal epithelial cells (IEC). UDP is the only endogenous P2Y6 receptor agonist. Previously, we showed that P2Y6R play a role in intestinal inflammation. In this study, we have investigated the role of P2Y6R in colorectal carcinogenesis. First, we have shown that invalidation of the P2ry6 gene expression in mice lead to significant reduction of the tumor load in the AOM-DSS murine model of CRC. Hence, we provided strong evidence that P2Y6R could play a role in tumor neovascularization and tumor cells proliferation by acting on Wnt/β-catenin signaling pathway. P2Y6R could have an anti-apoptotic function, and promote tumoral growth. The tumor promoting effect of P2Y6R might come from its ability to promote apoptosis resistance. Indeed, we showed that P2Y6R stimulation protected colorectal cancer cell HT-29 from TNF-induced apoptosis. This resistance could be induced by the stabilization of XIAP expression, an anti-apoptotic protein, through its phosphorylation. This resistance to cell death could be translated into increase resistance of tumoroids to 5-FU treatment. Secondly, we have shown that P2Y6R was involved in the integrity of epithelial barrier by promoting cytokine secretion involved in IEC migration, thus favouring the healing process, and by participating to the maintenance of intestinal mucus quality. Finally, we have shown that P2Y6R participated to neutrophils recruitment. Neutrophils are involved in innate immunity and can promote tumorigenesis by participating to the metastatic cascade and influencing angiogenesis. In summary, this study showed that P2Y6R could promote CRC tumorigenesis by modulating various mechanisms involved in colorectal carcinogenesis

Multidrug Resistance-Associated Protein 2 Expression Is Upregulated by Adenosine 5'-Triphosphate in Colorectal Cancer Cells and Enhances Their Survival to Chemotherapeutic Drugs.

Extracellular adenosine 5'-triphosphate (ATP) is a signaling molecule that induces a plethora of effects ranging from the regulation of cell proliferation to modulation of cancerous cell behavior. In colorectal cancer, ATP was reported to stimulate epithelial cell proliferation and possibly promote resistance to anti-cancer treatments. However, the exact role of this danger-signaling molecule on cancerous intestinal epithelial cells (IECs) in response to chemotherapeutic agents remains unknown. To address how ATP may influence the response of cancerous IECs to chemotherapeutic agents, we used Caco-2 cells, which display enterocyte-like features, to determine the effect of ATP on the expression of multidrug resistance-associated protein 2 (MRP2). Gene and protein expression were determined by quantitative real-time PCR (qRT-PCR) and Western blotting. Resistance to etoposide, cisplatin and doxorubicin was determined by MTT assays in response to ATP stimulation of Caco-2 cells and in cells for which MRP2 expression was down-regulated by shRNA. ATP increased the expression of MRP2 at both the mRNA and protein levels. MRP2 expression involved an ATP-dependent stimulation of the MEK/ERK signaling pathway that was associated with an increase in relative resistance of Caco-2 cells to etoposide. Abolition of MRP2 expression using shRNA significantly reduced the protective effect of MRP2 toward etoposide as well as to cisplatin and doxorubicin. This study describes the mechanism by which ATP may contribute to the chemoresistance of cancerous IECs in colorectal cancer. Given the heterogeneity of colorectal adenocarcinoma responses to anti-cancer drugs, these findings call for further study to understand the role of P2 receptors in cancer drug therapy and to develop novel therapies aimed at regulating P2 receptor activity

IC₅₀ and relative resistance (RR) values measured in response to etoposide, cisplatin and doxorubicin treatment of Caco-2 cells stably expressing non-target shRNA (shNT) or shMRP2.

<p>Caco-2 cells stably expressing shNT or shMRP2 (#305) were incubated with the cytotoxic drugs for 84 h. Sensitivity to the anti-cancer drug was determined by the MTT cell viability assay. Dose-response curves were fitted to the data to determine the toxicity (IC₅₀ value) of the drugs. Inhibition of human shMRP2 expression reduced the resistance of Caco-2 cells to etoposide, cisplatin and doxorubicin compared to control cells.</p><p>IC₅₀ and relative resistance (RR) values measured in response to etoposide, cisplatin and doxorubicin treatment of Caco-2 cells stably expressing non-target shRNA (shNT) or shMRP2.</p

The down-regulation of MRP2 expression by shRNA renders Caco-2 cells more sensitive to etoposide.

<p><b>(A)</b> Western blot analysis was used to assess the down-regulation of MRP2 protein expression in the presence of two shRNAs directed against the protein. Down-regulation was achieved by lentiviral infection of Caco-2 cells as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136080#pone.0136080.ref031" target="_blank">31</a>]. shRNA directed against MRP2 (sh305 and sh307) abolished protein expression by 90–100% comparatively to cells expressing a non-targeting shRNA (shNT). <b>(B)</b> Caco-2 cells stably expressing shNT or shMRP2 (#305) were incubated with the cytotoxic drug etoposide for 84 h. Sensitivity to the anti-cancer drug was determined by the MTT cell viability assay. A dose-response curve was fitted to the data to determine the toxicity (IC₅₀ value) of the drugs. The non-linear survival curves are presented as the mean ± SEM of four experiments performed in triplicate. Statistical significance was calculated using multiple <i>t</i>-test comparisons, where * p < 0.05 as compared with shNT. Inhibition of human shMRP2 expression reduced the resistance of Caco-2 cells to etoposide compared to control cells. IC₅₀ and RR values are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136080#pone.0136080.t001" target="_blank">Table 1</a>.</p

ATP-dependent stimulation of MRP2 protein expression is strongly reduced in the presence of the P2 receptor antagonist Suramin.

<p>Caco-2 cells were treated with 100 μM PPADS or Suramin 30 min prior to the addition of 100 μM ATP for 6 h. MRP2 expression was analyzed by Western blotting. ATP stimulated the expression of MRP2 as compared to non-stimulated (N-S) cells, whereas the addition of Suramin prior to the ATP stimulation strongly decreased MRP2 expression compared to ATP-stimulated cells in the presence of vehicle (DMSO (-)) only. The presented blot is typical of three separate sets of experiments.</p

MRP2 expression is regulated by MEK/ERK signaling cascade.

<p>Caco-2 cells were pretreated with NFκB (2 μM, Bay, BAY11-7082), MEK1/2 (10 μM, U0, U0126), PI3K (20 μM, LY, LY294003) and p38 (20 μM, SB, SB203580) inhibitors for 30 minutes and stimulated with 100 μM ATP for 6 h. <b>(A)</b> A typical Western blot against MRP2 is displayed from which <b>(B)</b> densitometry analysis showed a significant reduction in MRP2 expression in the presence of U0126, a selective MEK 1/2 inhibitor. Cells pretreated with U0126 led to a 75% reduction in the expression of MRP2 compared to ATP-stimulated cells only (-). Results are presented as the mean ± SEM of three separate experiments performed in duplicate. Statistical significance was determined by an unpaired <i>t</i>-test, where * p < 0.05 vs. non-stimulated (N-S) or ATP-stimulated cells as indicated on figure.</p

The G protein-coupled P2Y6 receptor promotes colorectal cancer tumorigenesis by inhibiting apoptosis

Colorectal tumors are immersed in an array of tumor-promoting factors including extracellular nucleotides such as uridine 5′‑diphosphate (UDP). UDP is the endogenous agonist of the G protein-coupled P2Y6 receptor (P2Y6R), which may contribute to the formation of a tumor-promoting microenvironment by coordinating resistance to apoptosis. Colorectal cancer (CRC) was chemically induced in P2ry6 knockout (P2ry6−/−) mice using azoxymethane and dextran sulfate sodium challenges. Mice were euthanatized and their tumor load determined. Fixed tissues were stained for histological and immunohistochemistry analysis. Tumoroids were also prepared from CRC tumors resected from P2ry6+/+ mice to determine the role of P2Y6R in resistance to apoptosis, whereas HT29 carcinoma cells were used to elucidate the signaling mechanism involved in P2Y6R anti-apoptotic effect. P2ry6−/− mice developed a reduced number of colorectal tumors with apparent tumors having smaller volumes. Overall dysplastic score was significantly lower in P2ry6−/− animals. Stimulation of P2Y6R with the selective agonist MRS2693 protected HT-29 cells from TNFα-induced apoptosis. This protective effect was mediated by the stabilizing phosphorylation of the X-linked inhibitor of apoptosis protein (XIAP) by AKT. Using CRC-derived tumoroids, P2Y6R activation was found to contribute to chemoresistance since addition of the P2Y6R agonist MRS2693 significantly prevented the cytotoxic effect of 5-fluorouracil. The present study shows that sustained activation of P2Y6R may contribute to intestinal tumorigenesis by blocking the apoptotic process and by contributing to chemoresistance, a substantial concern in the treatment of patients with CRC. These results suggest that P2Y6R may represent a prime target for reducing colorectal carcinogenesis.SCOPUS: ar.jinfo:eu-repo/semantics/publishe