La perte d'expression du récepteur de somatostatine sst2 accélère la cancérogenèse pancréatique induite par l'oncogène Kras : par quel(s) mécanisme(s) ?

L'adénocarcinome pancréatique canalaire (PDAC) reste un cancer mortel avec une incidence en progression et un pronostic sombre (4ème rang de cause de décès par cancer dans le monde). Les patients sont le plus souvent diagnostiqués à un stade métastatique où les thérapies actuelles restent inefficaces. Comprendre la physiopathologie de ce cancer permettra de proposer de nouveaux outils diagnostiques et thérapeutiques. Le développement de modèles transgéniques murins qui miment la pathologie humaine sera un atout majeur pour cette compréhension. En 2003, l'équipe d'Hingorani a développé des souris transgéniques où la mutation de l'oncogène Kras est spécifiquement introduite dans le pancréas (mutation présente dans 90% de PDAC). Ces souris (nommées KC) miment les étapes pré-néoplasiques mais ne développent que très rarement un cancer. Par ailleurs, les travaux de mon équipe ont montré que l'expression du récepteur à la somatostatine sst2, un récepteur couplé aux protéines G, est perdue dans 90% des cas de PDAC. En se fixant à son récepteur sst2, la somatostatine entraine une inhibition des sécrétions endocrines et exocrines pancréatiques, de la prolifération cellulaire, ou encore de l'inflammation. La réexpression de ce récepteur dans les cellules cancéreuses pancréatiques entraine un effet anti-tumoral par inhibition de l'axe PI3K/AKT/NF-?B. L'objectif de ma thèse était de montrer que le récepteur sst2 est un gène suppresseur de tumeur. Les souris invalidées pour le gène sst2 (sst2-/-) ne présentent pas de phénotype néoplasique pancréatique. J'ai donc entrepris leur croisement avec les souris KC. La caractérisation du modèle transgénique obtenu (souris Pdx1-KrasG12D/+ ; sst2+/-, nommé KCS) fait donc l'objet de mes travaux de thèse. Dès l'âge de 4 mois, les souris KCS développent des lésions cancéreuses alors que seules des lésions pré-néoplasiques sont observées chez les souris KC. Des analyses immunohistochimiques montrent que ces lésions cancéreuses se rapprochent phénotypiquement de celles observées dans la pathologie humaine (prolifération, métaplasie acino-canalaire, lésions de type canalaire de bas, moyen et haut grade, forte dysplasie nucléaire et cancer). Une activation plus soutenue de l'axe PI3K/AKT/NF-?B et de l'activité Kras est observée dans le pancréas des souris KCS par rapport aux KC. Ces lésions sont associées à une perte d'homéostasie du compartiment stromal (recrutement de fibroblastes, de cellules inflammatoires et immunitaires) suggérant que l'inflammation et la fibrose sont impliquées dans leur évolution. Les souris KCS présentent également une forte inflammation systémique, avec un ratio neutrophiles / lymphocytes élevé, en corrélation avec un pronostic sombre (moyenne de survie de 4 mois). Pour rechercher les médiateurs solubles sécrétés par ces lésions et impliquées dans leur genèse, une analyse exhaustive par " cytokines antibody array " des milieux conditionnés de primo-cultures d'acini pancréatiques isolés a permis l'identification de la chimiokine CXCL16 comme fortement et plus abondamment secrétée par les acini des souris KCS par rapport aux KC, et quantifiable dans les plasmas correspondants. Le traitement des souris KCS avec un anticorps bloquant CXCL16 empêche la forte activation de l'axe PI3K/AKT/NF-?B bloquant ainsi la carcinogenèse. De plus, CXCL16 et son récepteur CXCR6 sont surexprimés dans les lésions pancréatiques cancéreuses et acini environnants par rapport au pancréas normal murin ou humain. Nous proposons que le récepteur sst2 est un frein endogène limitant l'activation de l'axe PI3K/AKT/NF-?B. Sa perte d'expression amplifierait ces voies faiblement induites par la mutation seule de l'oncogène Kras, favorisant ainsi la carcinogenèse pancréatique via la mise en place d'une boucle d'amplification CXCL16-dépendante. Le ciblage de CXCL16 serait donc une stratégie thérapeutique prometteuse pour le PDAC, qui reste un des cancers les plus mortels.Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death. Its high mortality rate is primarily explained by its late diagnosis, aggressivity and resistance to therapies. Understanding the pathophysiology of this cancer will provide new diagnostic and therapeutic tools. The development of transgenic mouse models that mimic human disease will be a major asset to this understanding. In 2003, Hingorani team has developed transgenic mice that exhibit the conditional KrasG12D mutation in the pancreas (mutation present in 90% of PDAC). These mice (termed KC) mimic the pre-neoplastic stages but rarely develop cancer. Moreover, our team works show that expression of sst2 somatostatin receptor is lost in 90% of PDAC cases. Somatostatin binding to its receptor inhibits the endocrine and exocrine secretion, cell proliferation or inflammation. The re-expression of this receptor in pancreatic cancer cells leads to an antitumor effect by inhibiting the PI3K/AKT/NF-?B axis. My thesis objective demonstrates that the sst2 receptor is a tumor suppressor gene. The knockout mice for the sst2 gene (sst2-/-) show no neoplastic phenotype in the pancreas. I then took their intersection with the KC mice. The characterization of obtained transgenic model (Pdx1-KrasG12D/+ mice, sst2+/- named KCS) is thus the subject of my thesis work. We have developed a new transgenic mouse model named KCS to demonstrate that the sst2 receptor is a tumor suppressor gene whose expression loss in the presence of oncogenic Kras is important in the pancreatic cancer initiation, and to investigate the molecular and cellular events involved in this carcinogenesis. Phenotypic characterization of KCS mouse pancreas shows the occurrence as soon as 4 months of age of epithelial ductal lesions (acinar-to-ductal metaplasia, low- and high-grade of pancreatic intraepithelial neoplasia, high nuclear dysplasia, and cancer) that resemble those developed after 12 months by the KC model, and also developed in the human pathology. Interestingly, KCS mouse pancreatic lesions are associated with a loss of stromal homeostasis (recruitment of fibroblasts, inflammatory and immune cells, and fibrosis). These mice also exhibit a strong systemic inflammation, in correlation with a bad prognosis (median survival 4 months). Using membrane antibody arrays in isolated acini conditioned media allowed the identification of cytokines and chemokines involved in this strong intra-pancreatic inflammation. Thus, the chemokine CXCL16 is strongly secreted specifically by the acini and in plasmas of KCS mice. The neutralization of this chemokine, with antibody treatment, prevents PI3K/AKT/NF-?B hyperactivation, thereby blocking carcinogenesis. CXCL16 and its receptor CXCR6 are overexpressed in malignant lesions and surrounding acini as compared to normal pancreas in mouse or human tissues. We propose that sst2 expression loss and consequent relief of the physiological brake limiting PI3K/AKT/NF-kB activity amplifies Kras-driven pathways thus fostering pancreatic carcinogenesis through a CXCL16-dependent feedforward loop. Targeting CXCL16 may represent a promising therapeutic strategy for PDAC

Pharmacological targeting of the protein synthesis mTOR/4E-BP1 pathway in cancer-associated fibroblasts abrogates pancreatic tumourchemoresistance

International audiencePancreatic ductal adenocarcinoma (PDAC) is extremely stroma-rich. Cancer-associated fibroblasts (CAFs) secrete proteins that activate survival and promote chemoresistance of cancer cells. Our results demonstrate that CAF secretome-triggered chemoresistance is abolished upon inhibition of the protein synthesis mTOR/4E-BP1 regulatory pathway which we found highly activated in primary cultures of -SMA-positive CAFs, isolated from human PDAC resections. CAFs selectively express the sst1 somatostatin receptor. The SOM230 analogue (Pasireotide) activates the sst1 receptor and inhibits the mTOR/4E-BP1 pathway and the resultant synthesis of secreted proteins including IL-6. Consequently, tumour growth and chemoresistance in nude mice xenografted with pancreatic cancer cells and CAFs, or with pieces of resected human PDACs, are reduced when chemotherapy (gemcitabine) is combined with SOM230 treatment. While gemcitabine alone has marginal effects, SOM230 is permissive to gemcitabine-induced cancer cell apoptosis and acts as an antifibrotic agent. We propose that selective inhibition of CAF protein synthesis with sst1-directed pharmacological compounds represents an anti-stromal-targeted therapy with promising chemosensitization potential

Nucleolin Interacts and Co-Localizes with Components of Pre-Catalytic Spliceosome Complexes

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Multifaceted Nucleolin Protein and Its Molecular Partners in Oncogenesis

International audienceDiscovered in 1973, nucleolin is one of the most abundant phosphoproteins of the nucleolus. The ability of nucleolin to be involved in many cellular processes is probably related to its structural organization and its capability to form many different interactions with other proteins. Many functions of nucleolin affect cellular processes involved in oncogenesis-for instance: in ribosome biogenesis; in DNA repair, remodeling, and genome stability; in cell division and cell survival; in chemokine and growth factor signaling pathways; in angiogenesis and lymphangiogenesis; in epithelial-mesenchymal transition; and in stemness. In this review, we will describe the different functions of nucleolin in oncogenesis through its interaction with other proteins

Nucleolin Aptamer N6L Reprograms the Translational Machinery and Acts Synergistically with mTORi to Inhibit Pancreatic Cancer Proliferation

International audienceWe previously showed that N6L, a pseudopeptide that targets nucleolin, impairs pancreatic ductal adenocarcinoma (PDAC) growth and normalizes tumor vessels in animal models. In this study, we analyzed the translatome of PDAC cells treated with N6L to identify the pathways that were either repressed or activated. We observed a strong decrease in global protein synthesis. However, about 6% of the mRNAs were enriched in the polysomes. We identified a 5′TOP motif in many of these mRNAs and demonstrated that a chimeric RNA bearing a 5‘TOP motif was up-regulated by N6L. We demonstrated that N6L activates the mTOR pathway, which is required for the translation of these mRNAs. An inhibitory synergistic effect in PDAC cell lines, including patient-derived xenografts and tumor-derived organoids, was observed when N6L was combined with mTOR inhibitors. In conclusion, N6L reduces pancreatic cells proliferation, which then undergoes translational reprogramming through activation of the mTOR pathway. N6L and mTOR inhibitors act synergistically to inhibit the proliferation of PDAC and human PDX cell lines. This combotherapy of N6L and mTOR inhibitors could constitute a promising alternative to treat pancreatic cancer

Integrated analysis of mRNA and miRNA expression in HeLa cells expressing low levels of Nucleolin

International audienceNucleolin is an essential protein that plays important roles in the regulation of cell cycle and cell proliferation. Its expression is up regulated in many cancer cells but its molecular functions are not well characterized. Nucleolin is present in the nucleus where it regulates gene expression at the transcriptional and post-transcriptional levels. Using HeLa cells depleted in nucleolin we performed an mRNA and miRNA transcriptomics analysis to identify biological pathways involving nucleolin. Bioinformatic analysis strongly points to a role of nucleolin in lipid metabolism, and in many signaling pathways. Down regulation of nucleolin is associated with lower level of cholesterol while the amount of fatty acids is increased. This could be explained by the decreased and mis-localized expression of the transcription factor SREBP1 and the down-regulation of enzymes involved in the beta-oxidation and degradation of fatty acids. Functional classification of the miRNA-mRNA target genes revealed that deregulated miRNAs target genes involved in apoptosis, proliferation and signaling pathways. Several of these deregulated miRNAs have been shown to control lipid metabolism. This integrated transcriptomic analysis uncovers new unexpected roles for nucleolin in metabolic regulation and signaling pathways paving the way to better understand the global function of nucleolin within the cell

AS1411-conjugated gold nanoparticles affect cell proliferation through a mechanism that seems independent of nucleolin

International audienceG-rich oligonucleotide, AS1411, has been shown to interact with nucleolin and to inhibit cancer cell proliferation and tumor growth. This antiproliferative action is increased when AS1411 is conjugated to different types of nanoparticles. However, the molecular mechanisms are not known. In this work, we show in several cell lines that optimized AS1411-conjugated gold nanoparticles (GNS-AS1411) inhibit nucleolin expression at the RNA and protein levels. We observed an alteration of the nucleolar structure with a decrease of ribosomal RNA accumulation comparable to what is observed upon nucleolin knock down. However, the expression of genes involved in cell cycle and the cell cycle blockage by GNS-AS1411 are not regulated in the same way that in cells where nucleolin has been knocked down. These data suggest that the anti-proliferative activity of GNS-AS1411 is not the only consequence of nucleolin targeting and down-regulation