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Down-regulation of the epithelial cell-cell adhesion molecule E-cadherin is frequently associated with tumor formation and progression. Besides its role in physical cell-cell adhesion, E-cadherin is also thought to be involved in intracellular signaling in normal epithelial cells. In these cells, the Armadillo catenin p120ctn binds to the cytoplasmic domain of E-cadherin and stabilizes the adhesion complexes. On loss of E-cadherin, cytoplasmic p120ctn might accumulate and contribute to tumor malignancy. We used suppression subtractive hybridization to search for genes regulated by E-cadherin expression. We isolated human Nanos1 as a transcript of which levels decrease on E-cadherin reexpression in a human breast cancer cell line. The hNanos1 protein bears a COOH-terminal (CCHC)2 zinc finger domain and belongs to an evolutionarily conserved protein family sharing functions in germ cell development in both vertebrates and invertebrates. We found an inverse correlation between E-cadherin and hNanos1 expression in various cell lines and under diverse conditions. Conditional expression of hNanos1 in human colorectal DLD1 cancer cells functionally abolished cell-cell adhesion. It induced cytoplasmic translocation of p120ctn, as well as strong migratory and invasive properties. We also found that the NH2-terminal domain of hNanos1, which is conserved only among mammals, interacts with p120ctn. hNanos1 counteracted the stimulatory effect of p120ctn on cell protrusion formation. Together, these findings describe a new function for hNanos1 as a downstream effector of E-cadherin loss contributing to tumor progression. Targeting hNanos1 might be a promising strategy in the treatment of E-cadherin–negative tumors in particular. (Cancer Res 2006; 12(20): 10007-15)

Supplementary Figure 2 from E-Cadherin Regulates Human Nanos1, which Interacts with p120ctn and Induces Tumor Cell Migration and Invasion

Supplementary Methods, Materials and Figure Legends 1-2 from E-Cadherin Regulates Human Nanos1, which Interacts with p120ctn and Induces Tumor Cell Migration and Invasion

Supplementary Figure 1 from E-Cadherin Regulates Human Nanos1, which Interacts with p120ctn and Induces Tumor Cell Migration and Invasion

Background The regulation of mucociliary clearance is a key part of the defense mechanisms developed by the airway epithelium. If a high aggregate quality of evidence shows the clinical effectiveness of nasal irrigation, there is a lack of studies showing the intrinsic role of the different irrigation solutions allowing such results. This study investigated the impact of solutions with different pH and ionic compositions, eg, normal saline, non-diluted seawater and diluted seawater, on nasal mucosa functional parameters. Methods For this randomized, controlled, blinded, in vitro study, we used airway epithelial cells obtained from 13 nasal polyps explants to measure ciliary beat frequency (CBF) and epithelial wound repair speed (WRS) in response to 3 isotonic nasal irrigation solutions: (1) normal saline 0.9%; (2) non-diluted seawater (Physiomer®); and (3) 30% diluted seawater (Sterimar). The results were compared to control (cell culture medium). Results Non-diluted seawater enhanced the CBF and the WRS when compared to diluted seawater and to normal saline. When compared to the control, it significantly enhanced CBF and slightly, though nonsignificantly, improved the WRS. Interestingly, normal saline markedly reduced the number of epithelial cells and ciliated cells when compared to the control condition. Conclusion Our results suggest that the physicochemical features of the nasal wash solution is important because it determines the optimal conditions to enhance CBF and epithelial WRS thus preserving the respiratory mucosa in pathological conditions. Non-diluted seawater obtains the best results on CBF and WRS vs normal saline showing a deleterious effect on epithelial cell function.

LRP1 (low-density lipoprotein receptor-related protein 1), a multifunctional endocytic receptor, has recently been identified as a hub within a biomarker network for multi-cancer clinical outcome prediction. As its role in colon cancer has not yet been characterized, we here investigate the relationship between LRP1 and outcome. Materials and Methods LRP1 mRNA expression was determined in colon adenocarcinoma and paired colon mucosa samples, as well as in stromal and tumor cells obtained after laser capture microdissection. Clinical potential was further investigated by immunohistochemistry in a population-based colon cancer series (n = 307). LRP1 methylation, mutation and miR-205 expression were evaluated and compared with LRP1 expression levels. Results LRP1 mRNA levels were significantly lower in colon adenocarcinoma cells compared with colon mucosa and stromal cells obtained after laser capture microdissection. Low LRP1 immunohistochemical expression in adenocarcinomas was associated with higher age, right-sided tumor, loss of CDX2 expression, Annexin A10 expression, CIMP-H, MSI-H and BRAFV600E mutation. Low LRP1 expression correlated with poor clinical outcome, especially in stage IV patients. While LRP1 expression was downregulated by LRP1 mutation, LRP1 promoter was never methylated. Conclusions Loss of LRP1 expression is associated with worse colon cancer outcomes. Mechanistically, LRP1 mutation modulates LRP1 expression.

Introduction La bronchopneumopathie chronique obstructive (BPCO) est caracterisee par un remodelage de l’epithelium bronchique impliquant notamment les cellules ciliees. Etant donne l’existence d’anomalies intrinseques du processus de differenciation ciliee dans le contexte des ciliopathies, nous avons analyse la fonctionnalite et le processus de differentiation epithelial ciliaire a partir de cellules epitheliales bronchiques issues de patients atteints de BPCO et de sujets non BPCO. Methodes Les cellules epitheliales bronchiques obtenues par brossages cytologiques (n = 16 sujets BPCO, n = 16 sujets non BPCO) ont ete analysees dans les 30 minutes suivant le prelevement par videomicroscopie. La frequence ciliaire a ete mesuree a l’aide de la methode automatisee et standardisee developpee dans le plugin CiliaFA. Les cellules epitheliales bronchiques de 10 sujets BPCO et 10 sujets non BPCO ont ete cultivees a l’interface air-liquide, puis immunomarquees pour detecter et quantifier les cellules ciliees a 21 et 35 jours de differenciation. Les analyses statistiques ont ete realisees a l’aide des tests non-parametriques de Wilcoxon et Kruskal–Wallis au seuil alpha de 5 % en bilateral. Resultats Comparativement au groupe controle, les sujets BPCO presentaient une diminution de la frequence ciliaire (11,2 ± 3,3 vs 7,9 ± 3,3 Hz respectivement, p = 0,03) et une diminution de 50 % du nombre de cellules ciliees apres 21 jours (p = 0,005) et 35 jours de differentiation (p = 0,002). De plus, le nombre de cellules ciliees en fin de differenciation dans les cultures issues de patients BPCO n’atteignaient toujours pas celui des cultures de reference apres 21 jours d’IAL, suggerant une cinetique de differenciation ciliee alteree au cours de la BPCO. Conclusion Cette etude demontre une reduction de la frequence ciliaire epitheliale bronchique et suggere un retard dans le processus de differenciation ciliee au cours de la BPCO. Ces alterations de la fonction ciliaire motrice et du processus de differenciation ciliaire suggerent l’implication d’une ciliopathie propre a la BPCO, dont les mecanismes physiopathologiques et les eventuelles possibilites de strategies therapeutiques correctrices restent a elucider.

International audience; Chronic inflammation is a hallmark of cystic fibrosis (CF) lung disease and airway epithelium damage and remodelling are important components of lung pathology progression in CF. Whether this remodelling is secondary to deleterious infectious and inflammatory mediators, or to alterations of CF human airway epithelial (HAE) cells, such as their hyper inflammatory phenotype or their basic cystic fibrosis transmembrane conductance regulator (CFTR) default, remains debated. In this study, we evaluated the involvement of alterations of CF HAE cells in airway epithelium remodelling. HAE cells from non‐CF and CF patients were cultured in an air–liquid interface, with and without inflammatory stimulation, along the regeneration process, and the remodelling of the reconstituted epithelium was analysed. We confirmed that CF HAE cells showed a hyperinflammatory phenotype which was lost with time. In comparison to non‐CF epithelium, CF epithelium regeneration in the absence of exogenous inflammation was higher and exhibited basal cell hyperplasia. This remodelling was mimicked by inflammatory stimulation of non‐CF cells and was absent when CF HAE cells were no longer hyperinflamed. Moreover, the number of goblet cells was similar in non‐CF and CF cultures and increased equally under inflammatory stimulation. Finally, whatever the inflammatory environment, CF cultures showed a delay in ciliated cell differentiation. In conclusion, alterations of CF HAE cells partly regulate airway epithelium remodelling following injury and regeneration. This remodelling, together with goblet cell hyperplasia induced by exogenous inflammation and alteration of ciliated cell differentiation, may worsen mucociliary clearance impairment, leading to injury

In different physiopathological situations such as embryogenesis, wound repair and tumor invasion, isolated cells, or cell populations exhibit changes to their normal behavior and may acquire different migratory phenotypes. Live-cell imaging associated with the use of appropriate in vitro models in culture has become a powerful analytical tool for studying factors involved in cell migration and in cell-to-cell interactions. The scope of this chapter is to give an overview of in vitro models of cell migration and the technical advances permitting multiparameter quantification. The chapter hereby provides a detailed description of two-dimensional and three-dimensional approaches of cell dispersion and migration and finally gives a brief insight on computational quantification of the cell behavior.

In different physiopathological situations such as embryogenesis, wound repair and tumor invasion, isolated cells, or cell populations exhibit changes to their normal behavior and may acquire different migratory phenotypes. Live-cell imaging associated with the use of appropriate in vitro models in culture has become a powerful analytical tool for studying factors involved in cell migration and in cell-to-cell interactions. The scope of this chapter is to give an overview of in vitro models of cell migration and the technical advances permitting multiparameter quantification. The chapter hereby provides a detailed description of two-dimensional and three-dimensional approaches of cell dispersion and migration and finally gives a brief insight on computational quantification of the cell behavior.

Epithelial-to-mesenchymal transition (EMT) phenomena endow epithelial cells with enhanced migratory and invasive potential, and as such, have been implicated in many physiological and pathological processes requiring cell migration/invasion. Although their involvement in the metastatic cascade is still a subject of debate, data are accumulating to demonstrate the existence of EMT phenotypes in primary human tumors, describe enhanced metastatic potential of EMT derivatives in animal models, and report EMT attributes in circulating tumor cells (CTCs). The relationships between EMT and CTCs remain largely unexplored, and we review here in vitro and in vivo data supporting a putative role of EMT processes in CTC generation and survival.

International audience; In many types of cancers, the fragile histidine triad (Fhit) gene is frequently targeted by genomic alterations leading to a decrease or loss of gene and protein expression. Fhit has been described as a tumor suppressor gene because of its ability to induce apoptosis and to inhibit proliferation of tumor cells. Moreover, several studies have shown a correlation between the lack of Fhit expression and tumor aggressiveness, thus suggesting that Fhit could be involved in tumor progression. In this study, we explored the potential role of Fhit during tumor cell invasion. We first showed that a low Fhit expression is associated with in vivo and in vitro invasiveness of tumor cells. Then, we showed that Fhit overexpression in Fhit-negative highly invasive NCI-H1299 cells by transfection of Fhit cDNA and Fhit inhibition in Fhit-positive poorly invasive HBE4-E6/E7 cells by transfection of Fhit small interfering RNA induce, respectively, a decrease and an increase in migratory/invasive capacities. These changes in cell behavior were associated with a reorganization of tight and adherens junction molecules and a regulation of matrix metalloproteinase and vimentin expression. These results show that Fhit controls the invasive phenotype of lung tumor cells by regulating the expression of genes associated with epithelial-mesenchymal transition.Oncogene advance online publication, 23 November 2009; doi:10.1038/onc.2009.418.

Background Many studies associated the main polyphenolic constituent of green tea, (-)-Epigallocatechin-3-gallate (EGCG), with inhibition of cancers, invasion and metastasis. To date, most of the studies have focused on the effect of EGCG on cell proliferation or death. Since cell migration is an important mechanism involved in tumor invasion, the aim of the present work was to target another approach of the therapeutic effect of EGCG, by investigating its effect on the cell migratory behavior. Methods The effect of EGCG (at concentrations lower than 10 μg/ml) on the migration speed of invasive cells was assessed by using 2D and 3D models of cell culture. We also studied the effects of EGCG on proteinases expression by RT-PCR analysis. By immunocytochemistry, we analyzed alterations of vimentin organization in presence of different concentrations of EGCG. Results We observed that EGCG had an inhibitory effect of cell migration in 2D and 3D cell culture models. EGCG also inhibited MMP-2 mRNA and protein expression and altered the intermediate filaments of vimentin. Conclusion Taken together, our results demonstrate that EGCG is able to inhibit the migration of bronchial tumor cells and could therefore be an attractive candidate to treat tumor invasion and cell migration.

Nicotinic acetylcholine receptors (nAChRs), present in human bronchial epithelial cells (HBECs), have been shown in vitro to modulate cell shape. Because cell spreading and migration are important mechanisms involved in the repair of the bronchial epithelium, we investigated the potential role of nAChRs in the wound repair of the bronchial epithelium. In vivo and in vitro, alpha3alpha5beta2-nAChRs accumulated in migrating HBECs involved in repairing a wound, whereas alpha7-nAChRs were predominantly observed in stationary confluent cells. Wound repair was improved in the presence of nAChR agonists, nicotine, and acetylcholine, and delayed in the presence of alpha3beta2 neuronal nAChR antagonists, mecamylamine, alpha-conotoxin MII, and kappa-bungarotoxin; alpha-bungarotoxin, an antagonist of alpha7-nAChR, had no effect. Addition of nicotine to a repairing wound resulted in a dose-dependent transient increase of intracellular calcium in migrating cells that line the wound edge. Mecamylamine and kappa-bungarotoxin inhibited both the cell-migration speed and the nicotine-induced intracellular calcium increase in wound-repairing migrating cells in vitro. On the contrary alpha-bungarotoxin had no significant effect on migrating cells. These results suggest that alpha3alpha5beta2-nAChRs actively contribute to the wound repair process of the respiratory epithelium by modulating intracellular calcium in wound-repairing migrating cells.

Down-regulation of the epithelial cell-cell adhesion molecule E-cadherin is frequently associated with tumor formation and progression. Besides its role in physical cell-cell adhesion, E-cadherin is also thought to be involved in intracellular signaling in normal epithelial cells. In these cells, the Armadillo catenin p120ctn binds to the cytoplasmic domain of E-cadherin and stabilizes the adhesion complexes. On loss of E-cadherin, cytoplasmic p120ctn might accumulate and contribute to tumor malignancy. We used suppression subtractive hybridization to search for genes regulated by E-cadherin expression. We isolated human Nanos1 as a transcript of which levels decrease on E-cadherin reexpression in a human breast cancer cell line. The hNanos1 protein bears a COOH-terminal (CCHC)2 zinc finger domain and belongs to an evolutionarily conserved protein family sharing functions in germ cell development in both vertebrates and invertebrates. We found an inverse correlation between E-cadherin and hNanos1 expression in various cell lines and under diverse conditions. Conditional expression of hNanos1 in human colorectal DLD1 cancer cells functionally abolished cell-cell adhesion. It induced cytoplasmic translocation of p120ctn, as well as strong migratory and invasive properties. We also found that the NH2-terminal domain of hNanos1, which is conserved only among mammals, interacts with p120ctn. hNanos1 counteracted the stimulatory effect of p120ctn on cell protrusion formation. Together, these findings describe a new function for hNanos1 as a downstream effector of E-cadherin loss contributing to tumor progression. Targeting hNanos1 might be a promising strategy in the treatment of E-cadherin–negative tumors in particular. (Cancer Res 2006; 12(20): 10007-15)

Introduction L’invasion tumorale est un processus complexe qui requiert des modifications des proprietes d’adherence et un remaniement de la matrice extracellulaire. De nombreuses etudes ont montre une correlation entre la reorganisation du complexe cadherine E/catenines (molecules impliquees dans les liens intercellulaires) et l’expression accrue d’enzymes proteolytiques de la famille des metalloproteinases matricielles (MMPs). Il a ete montre recemment que hNanosl, molecule reprimee par la cadherine E, est impliquee dans l’acquisition d’un phenotype infiltrant par les cellules tumorales. Le but de cette etude est donc d’evaluer le role potentiel de hNanosl sur l’expression des MMPs par les cellules tumorales. Methodes Dans un premier temps, nous avons transfecte le vecteur d’expression de hNanosl dans des cellules tumorales non invasives et des siRNAs diriges contre hNanosl dans des cellules tumorales invasives. Nous avons analyse l’expression de MMPs dans les diverses cellules tumorales transfectees par western blotting, zymographie et RT-PCR. Nous avons egalement mesure la capacite infiltrante de ces cellules transfectees en test de migration en chambre de Boyden. Nous avons enfin realise une etude in vivo de l’expression de hNanosl et des MMPs dans des tissus bronchiques normaux et tumoraux par RT-PCR. Resultats Nous avons observe que hNanos 1 augmente l’expression de la MMP-2 et de la MT1-MMP dans les cellules tumorales non invasives ainsi que leur potentiel infiltrant. A l’inverse, l’inhibition de son expression par des siRNAs diminue l’expression de ces deux enzymes et la capacite infiltrante des cellules tumorales. Enfin, notre etude in vivo a revele que hNanosl est surexprime dans les tissus tumoraux par rapport aux tissus normaux. L’expression de hNanosl est correlee a l’expression de la MMP-2 et de la MT1-MMP dans les tissus tumoraux. Conclusion Ces resultats demontrent que hNanosl joue un role dans l’acquisition de proprietes infiltrantes par les cellules tumorales en agissant sur l’expression de MMPs. Ceci met en evidence l’existence d’un nouveau mecanisme implique dans le processus d’invasion tumorale.

Introduction Les recepteurs nicotiniques a l’acetylcholine sont exprimes au sein de l’epithelium bronchique et sont impliques in vitro dans le maintien de la forme cellulaire (Res Commun Mol Pathol Pharmacol 1997 ; 97 : 243-62). L’etalement et la migration cellulaires sont des mecanismes importants du processus normal du remodelage bronchique. Nous nous sommes interesses au role potentiel des recepteurs nicotiniques dans la reparation de l’epithelium respiratoire humain. Methodes Des tissus bronchiques humains (modele ex vivo de lesion/reparation) et des cultures primaires de cellules bronchiques humaines (modele in vitro de lesion/reparation) ont ete localement leses et le processus de reparation, la migration cellulaire et les variations de calcium intracellulaire ont ete etudies par des techniques de video-microscopie, en presence d’antagonistes specifiques des recepteurs nicotiniques α3β2 (mecamylamine, a-conotoxine Mil et κ-bungarotoxine) ou des recepteurs a7 (a-bungarotoxine). L’expression et la distribution des recepteurs nicotiniques ont ete etudiees par des techniques d’immunofluorescence et de RT-PCR. Resultats Ex vivo et in vitro, les sous-unites α3, α5 et P2 des recepteurs nicotiniques sont surexprimees dans les cellules bronchiques qui migrent pour reparer la lesion, alors que la sous-unite a7 est essentiellement observee dans les cellules stationnaires. La vitesse de reparation des lesions est augmentee en presence de nicotine et d’acetylcholine et diminuee en presence de mecamylamine, d’a-conotoxine Mil et de κ-bungarotoxine, mais inchangee en presence d’a-bungarotoxine. L’addition de nicotine in vitro pendant la reparation de lesions induit une augmentation transitoire du calcium intracellulaire libre dans les cellules migratoires qui bordent la lesion. La migration cellulaire et l’augmentation de calcium intracellulaire induite par la nicotine sont inhibees par la mecamylamine et la κ-bungarotoxine, mais inchangees en presence d’α-bungarotoxine. Conclusion Ces resultats suggerent que le recepteur nicotinique α3β2 contribue activement a la reparation epitheliale en modulant la migration cellulaire et les concentrations calciques intracellulaires dans les cellules migratoires situees en bordure de la lesion.