Coordinate-based co-localization-mediated analysis of arrestin clustering upon stimulation of the C-C chemokine receptor 5 with RANTES/CCL5 analogues

G protein-coupled receptor activation and desensitization leads to recruitment of arrestin proteins from cytosolic pools to the cell membrane where they form clusters difficult to characterize due to their small size and further mediate receptor internalization. We quantitatively investigated clustering of arrestin 3 induced by potent anti-HIV analogues of the chemokine RANTES after stimulation of the C-C chemokine receptor 5 using single-molecule localization-based super-resolution microscopy. We determined arrestin 3 cluster sizes and relative fractions of arrestin 3 molecules in each cluster through image-based analysis of the localization data by adapting a method originally developed for co-localization analysis from molecular coordinates. We found that only classical agonists in the set of tested ligands were able to efficiently recruit arrestin 3 to clusters mostly larger than 150nm in size and compare our results with existing data on arrestin 2 clustering induced by the same chemokine analogues

The transcription factor FOXM1 regulates the balance between proliferation and aberrant differentiation in head and neck squamous cell carcinoma

Sustained expression of FOXM1 is a hallmark of nearly all human cancers including squamous cell carcinomas of the head and neck (HNSCC). HNSCCs partially preserve the epithelial differentiation program, which recapitulates fetal and adult traits of the tissue of tumor origin but is deregulated by genetic alterations and tumor-supporting pathways. Using shRNA-mediated knockdown, we demonstrate a minimal impact of FOXM1 on proliferation and migration of HNSCC cell lines under standard cell culture conditions. However, FOXM1 knockdown in three-dimensional (3D) culture and xenograft tumor models resulted in reduced proliferation, decreased invasion, and a more differentiated-like phenotype, indicating a context-dependent modulation of FOXM1 activity in HNSCC cells. By ectopic overexpression of FOXM1 in HNSCC cell lines, we demonstrate a reduced expression of cutaneous-type keratin K1 and involucrin as a marker of squamous differentiation, supporting the role of FOXM1 in modulation of aberrant differentiation in HNSCC. Thus, our data provide a strong rationale for targeting FOXM1 in HNSCC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Rôle of the discoidin domain receptor tyrosine kinase 2 in head and neck cancer-associated fibroblasts

Malgré de nombreuses avancées sur les traitements et le diagnostic des carcinomes épidermoïdes de la tête et du cou (head and neck squamous cell carcinoma - HNSCC), le taux de survie des patients n'a pas beaucoup augmenté ces 20 dernières années. Suite au traitement, l'agressivité des tumeurs récurrentes est souvent liée au manque de contrôle local ou locorégional de la tumeur. Ainsi, la résection chirurgicale des tumeurs est souvent complémentée par de la chimiothérapie ou de la radio-chimiothérapie visant les cellules cancéreuses disséminées en dehors des marges négatives de la tumeur. Afin d'améliorer les traitements existants et de diminuer les risques de récurrences et de métastases, il est donc primordial de comprendre le mécanisme d'invasion des cellules cancéreuses en dehors des marges chirurgicales. L'invasion des cellules tumorales est notamment régulée par le microenvironnement de la tumeur qui est en grande partie constitué de fibroblastes associés au cancer [cancer-associated fibroblasts - CAFs). Les CAFs jouent un rôle critique dans l'invasion des cellules tumorales, notamment en sécrétant un grand nombre de facteurs pro-tumorigéniques. Cette population de cellule est très hétérogène en raison du fait qu'elles proviennent de différents précurseurs en cours de différentiation fibrotique ou myofibroblastique. Quelle que soit leur origine, les CAFs ont néanmoins tous en commun l'expression d'un récepteur au collagène connu sous le nom de récepteur à domaine discoïdine 2, DDR2. Il a récemment été montré que l'expression de ce récepteur est impliquée dans les activités pro-métastatique et pro-invasive de différents types de cancers, tels que les cancers de la tête et du cou. En raison de son expression dans les CAFs et dans les cellules en transition épithélio-mésenchymateuse, DDR2 constitue donc une cible intéressante pour le développement de médicaments anticancéreux. Afin de mieux comprendre le rôle de DDR2 dans les fibroblastes du cancer HNSCC, nous avons isolé plusieurs lignées de fibroblastes provenant de différents tissus de la tête et du cou. La fonctionnalité de DDR2 dans les CAFs a été étudiée à l'aide de modèles cellulaires de surexpression ou de réduction d'expression de DDR2. Nous avons notamment pu démontrer que DDR2 est primordial pour la prolifération des CAFs, ainsi que pour la sécrétion de facteurs pro-tumorigéniques comme HGF et POSTN. Par des modèles de co- cultures organotypiques, nous avons pu démontrer que l'expression de DDR2 au sein des CAFs module l'invasion des cellules cancéreuses. En utilisant un modèle murin de chirurgie orthotopique basé sur la co- implantation de CAF humains avec des cellules HNSCC, nous avons mis en évidence l'impact de l'expression de DDR2 dans les CAFs sur le phénotype tumoral. En effet, son expression dans les CAFs induit une augmentation de l'invasion des cellules cancéreuses dans les vaisseaux lymphatiques, ainsi qu'une augmentation de la formation des vaisseaux lymphatiques. Dans l'ensemble, cette étude apporte une preuve supplémentaire de l'importance de DDR2 en tant que cible thérapeutique prometteuse dans le cancer de la tête et du cou. -- Despite recent advances in diagnostics and therapy, head and neck squamous cell carcinoma (HNSCC) is still deadly because of treatment failures affecting a significant numberof patients. Poor control at local and at loco-regional sites are frequent causes of aggressive tumor récurrences arising after treatment. Surgical resection is a pivotai treatment aiming to completely remove tumors. However, the high risk of cancer cell dissémination outside of the tumor-negative surgical margins nécessitâtes adjuvant chemo- or chemoradio- therapy. Understanding of the mechanisms underlying cancer cell spread beyond the surgical margins is a necessary step to establish therapeutic approaches aiming to prevent récurrences and métastasés. Invasive phenotype of cancer cells is controlled by their microenvironment, where cancer-associated fibroblasts (CAFs) are the most critical cells. CAFs can secrete a large number of pro-tumorigenic factors and remodel the extracellular matrix. They are highly heterogeneous cells originating from a variety of mesenchymal precursors undergoing fibrotic or myofibroblastic differentiation and sharing many common features such as the collagen receptor DDR2. Recently, evidence emerged for pro-invasive and -metastatic activity of DDR2 in différent cancer types, including HNSCC, thus providing an attractive target expressed by CAFs and cancer cells undergoing epithelial-mesenchymal transition. To better understand the rôle of fibroblasts in HNSCC, we established multiple lines of fibroblasts from the head and neck cancer and normal tissue that included normal fibroblasts (NF), fibroblasts from the tumor (.cancer-associated fibroblasts - CAF), and fibroblasts from the matched tumor-free margins (MF). In this collection of fibroblasts, we assessed the expression of known marker of CAF phenotype and performed functional assays. Focusing on the functionality of DDR2 in CAFs, we established cellular models, including organotypic co-cultures, for DDR2 silencing and overexpression to address the DDR2-mediated rôle of CAFs in cancer cell invasion. We provided evidences for a rôle of DDR2 in prolifération of fibroblasts and sécrétion of a number of pro-invasive factors, such as periostin and HGF. We found that DDR2 pathway may employ both kinase-dependent and -independent mechanisms to control phenotype of fibroblasts, and, like in cancer cells, DDR2 pathway regulates expression of Snaill, a transcriptional regulator of fibroblast-specific genes. Usingan orthotopic surgical mouse model based on co-implantation of human CAFs (DDR2-silenced or DDR2-overexpressing) with HNSCC cells, we highlighted the impact of the fibroblastic DDR2 on tumor phenotype. CAFs overexpressing DDR2 induced the increased spread of cancer cells in lymphatic vessels along with the increased lymphatic vessel formation. Overall, this study provides further support for DDR2 as a promising therapeutic target in head and neck cancer

Quantitative morphological analysis of arrestin2 clustering upon G protein-coupled receptor stimulation by super-resolution microscopy

Clustering of arrestins upon G protein-coupled receptor stimulation is a phenomenon that is well-known but difficult to describe quantitatively due to the size of the clusters close to the diffraction limit of visible light. We introduce a general method to quantitatively investigate the clustering of arrestin following stimulation of the C-C chemokine receptor 5 (CCR5) using single-molecule super-resolution imaging and coordinate and image-based cluster analysis. We investigated the effect of potent anti-HIV ligands of CCR5 with different pharmacological profiles on arrestin2 cluster formation and found that only the ligands capable of inducing CCR5 internalization induced arrestin2 recruitment and clustering. We further demonstrate that the fraction of arrestin2 molecules found in clusters larger than 100nm correlates with the magnitude of ligand-induced CCR5 internalization, but not with G protein activation, indicating that recruitment of arrestin2 to CCR5 is independent of G protein activation. Pre-treatment of the cells with the drug cytochalasin D, which blocks actin polymerization, led to the formation of larger clusters, whereas the inhibitor of microtubule polymerization nocodazole had little effect on arrestin2 recruitment, suggesting an active role of actin in the organization and dynamics of these aggregates

Coordinate-based co-localization-mediated analysis of arrestin clustering upon stimulation of the C-C chemokine receptor 5 with RANTES/CCL5 analogues

G protein-coupled receptor activation and desensitization leads to recruitment of arrestin proteins from cytosolic pools to the cell membrane where they form clusters difficult to characterize due to their small size and further mediate receptor internalization. We quantitatively investigated clustering of arrestin 3 induced by potent anti-HIV analogues of the chemokine RANTES after stimulation of the C-C chemokine receptor 5 using single-molecule localization-based super-resolution microscopy. We determined arrestin 3 cluster sizes and relative fractions of arrestin 3 molecules in each cluster through image-based analysis of the localization data by adapting a method originally developed for co-localization analysis from molecular coordinates. We found that only classical agonists in the set of tested ligands were able to efficiently recruit arrestin 3 to clusters mostly larger than 150 nm in size and compare our results with existing data on arrestin 2 clustering induced by the same chemokine analogues