The appearance of hybrid epithelial-mesenchymal (E/M) cells expressing E-cadherin is favourable for the establishment of pro-invasive function. Although the potential role of E-cadherin in cancer invasion is now accepted, the molecular mechanisms involved in this process are not completely elucidated. To gain further insight, we focused our analysis on invadopodia formation, an early event in the invasion process. We used models of E/M hybrid cell lines, tissue sections and patient-derived xenografts from a multi-centre clinical trial. E-cadherin involvement in invadopodia formation was assessed using a gelatin-FITC degradation assay. Mechanistic studies were performed by using proteomic analysis, siRNA strategy and proximity ligation assay. We showed that E-cadherin is a critical component of invadopodia. This unexpected localization results from a synergistic trafficking of E-cadherin and MT1-MMP through a Rab vesicle-dependent pathway. Modulation of E-cadherin expression or activation impacted invadopodia formation. Moreover, colocalization of E-cadherin and Actin in "ring structures" as precursors of invadopodia reveals that E-cadherin is required for invadopodia structuration. E-cadherin, initially localised in the adherens junctions, could be recycled to nascent invadopodia where it will interact with several components enriched in invadopodia, such as Arp2/3, Cortactin or MT1-MMP. The trans-adhesive properties of E-cadherin are therefore essential for structuring invadopodia. This new localisation of E-cadherin and its unexpected role in cell invasion shine a new light on hybrid E/M transition features in tumoral invasion.

Supplementary Materials, Legends for Table 1 and Figures 1-8 from ArgBP2-Dependent Signaling Regulates Pancreatic Cell Migration, Adhesion, and Tumorigenicity

Graphical abstract Background The appearance of hybrid epithelial-mesenchymal (E/M) cells expressing E-cadherin is favourable for the establishment of pro-invasive function. However, the molecular mechanism and potential roles of E-cadherin in cancer cell invasion stay unexplored. Methods We used models of E/M hybrid cell lines, tissues sections and patient-derived xenografts from a multi-center clinical trial. E-cadherin involvement in invadopodia formation was assessed using a gelatin-FITC degradation assay. Mechanistic studies were performed by using proteomic analysis, siRNA strategy and proximity ligation assay. Results We showed that E-cadherin is a critical component of invadopodia. This unexpected localization results from a synergistic trafficking of E-cadherin and MT1-MMP through Rab vesicle-dependent pathway. Modulation of E-cadherin expression or activation impacted invadopodia formation. Moreover, colocalization of E-cadherin and Actin in ―ring structures‖ as precursor of invadopodia reveals that E-cadherin is required for invadopodia structuration. Conclusion E-cadherin, initially localized in the adherens junctions could be recycled to nascent invadopodia where it will interact with several components such as Arp2/3, Cortactin or MT1-MMP. The trans-adhesive properties of E-cadherin are therefore essential for structuring invadopodia .

Intestinal barrier defects lead to “leaky gut syndrome”, defined as an increase in intestinal permeability that allows the passage of luminal content into intestinal tissue and the bloodstream. Such a compromised intestinal barrier is the main factor underlying the pathogenesis of inflammatory bowel disease, but also commonly occurs in various systemic diseases such as viral infections and metabolic syndrome. The non-pathogenic yeast Saccharomyces boulardii CNCM I-745 has demonstrated its effectiveness as a probiotic in the prevention and treatment of antibiotic-associated, infectious and functional diarrhea. Via multiple mechanisms of action implicated in intestinal barrier function, S. boulardii has beneficial effects on altered intestinal microbiota and epithelial barrier defects in different pathologies. The well-studied probiotic yeast S. boulardii plays a crucial role in the preservation and/or restoration of intestinal barrier function in multiple disorders. This could be of major interest in diseases characterized by alterations in intestinal barrier function.

Cadherins are a large family of transmembrane calcium-dependent cell adhesion proteins that orchestrate adherens junction formation and are crucially involved in tissue morphogenesis. Due to their important role in cancer development and metastasis, cadherins can be considered attractive targets for drug discovery. A recent crystal structure of the complex of a cadherin extracellular portion and a small molecule inhibitor allowed the identification of a druggable interface, thus providing a viable strategy for the design of cadherin dimerization modulators. Here, we report on a structure-based virtual screening approach that led to the identification of efficient and selective modulators of E-cadherin-mediated cell cell adhesion. Of all the putative inhibitors that were identified and experimentally tested by cell adhesion assays using human pancreatic tumor BxPC-3 cells expressing both E-cadherin and P-cadherin, two compounds turned out to be effective in inhibiting stable cell-cell adhesion at micromolar concentrations. Moreover, at the same concentrations, one of them also showed anti-invasive properties in cell invasion assays. These results will allow further development of novel and selective cadherin-mediated cell cell adhesion modulators for the treatment of a variety of cadherin-expressing solid tumors and for improving the efficiency of drug delivery across biological barriers.

Pancreatic adenocarcinoma (PAC) has a poor prognosis. One treatment approach, investigated here, is to reinforce antitumor immunity. Dendritic cells (DCs) are essential for the development and regulation of adaptive host immune responses against tumors. A major role for DCs may be as innate tumoricidal effector cells. We explored the efficacy of vaccination with immature (i)DCs, after selecting optimal conditions for generating immunostimulatory iDCs. We used two models, C57BL/6Jrj mice with ectopic tumors induced by the PAC cell line, Panc02, and genetically engineered (KIC) mice developing PAC. Therapeutic iDC-vaccination resulted in a significant reduction in tumor growth in C57BL/6Jrj mice and prolonged survival in KIC mice. Prophylactic iDC-vaccination prevented subcutaneous tumor development. These protective effects were long-lasting in Panc02-induced tumor development, but not in melanoma. iDC-vaccination impacted the immune status of the hosts by greatly increasing the percentage of CD8+ T-cells, and natural killer (NK)1.1+ cells, that express granzyme B associated with Lamp-1 and IFN-γ. Efficacy of iDC-vaccination was CD8+ T-cell-dependent but NK1.1+ cell-independent. We demonstrated the ability of DCs to produce peroxynitrites and to kill tumor cells; this killing activity involved peroxynitrites. Altogether, these findings make killer DCs the pivotal actors in the beneficial clinical outcome that accompanies antitumor immune responses. We asked whether efficacy can be improved by combining DC-vaccination with the FOLFIRINOX regimen. Combined treatment significantly increased the lifespan of KIC mice with PAC. Prolonged treatment with FOLFIRINOX clearly augmented this beneficial effect. Combining iDC-vaccination with FOLFIRINOX may therefore represent a promising therapeutic option for patients with PAC.

BACKGROUND:Pancreatic ductal adenocarcinoma (PDAC) is characterised by an extensive tissue invasion and an early formation of metastasis. Alterations in the expression of cadherins have been reported in PDAC. Yet, how these changes contribute to tumour progression is poorly understood. Here, we investigated the relationship between cadherins expression and PDAC development.METHODS:Cadherins expression was assessed by immunostaining in both human and murine tissue specimens. We have generated pancreatic cancer cell lines expressing both cadherin-1 and cadherin-3 or only one of these cadherins. Functional implications of such genetic alterations were analysed both in vitro and in vivo.RESULTS:Cadherin-3 is detected early at the plasma membrane during progression of pancreatic intraepithelial neoplasia 1 (PanIN-1) to PDAC. Despite tumoural cells turn on cadherin-3, a significant amount of cadherin-1 remains expressed at the cell surface during tumourigenesis. We found that cadherin-3 regulates tumour growth, while cadherin-1 drives type I collagen organisation in the tumour. In vitro assays showed that cadherins differentially participate to PDAC aggressiveness. Cadherin-3 regulates cell migration, whereas cadherin-1 takes part in the invadopodia activity.CONCLUSIONS:Our results show differential, but complementary, roles for cadherins during PDAC carcinogenesis and illustrate how their expression conditions the PDAC aggressiveness.

Our understanding of the determinants of quality of life (QOL) in people living with Parkinson's disease and related disorders (PDRD) has grown remarkably in the past decade. However, several areas remain understudied including determinants of general vs. health-related QOL, determinants in high-need patients, drivers of perceptions of caregivers vs. patients, and exploration of potential determinants outside of the traditional medical model.This was a cross-sectional study of 210 PDRD patients and 175 caregivers who completed a battery of measures regarding general QOL (QOL-Alzheimer's disease; QOL-AD), health-related QOL (Parkinson's disease Questionnaire; PDQ-39), cognitive function, mood, grief, spiritual wellbeing, symptom burden, disease severity, disease stage, overall function, socioeconomic status, and healthcare utilization. Elastic net regularization modeling of variables significantly associated with our outcomes of interest were performed to determine predictors of general QOL, compare predictors of general vs. health-related QOL, and compare predictors of patient and caregiver perspectives on patient general QOL.General QOL was associated with spiritual wellbeing, depression, cognitive function, presence of a caregiver, and recent emergency department visits. In contrast, health-related QOL was associated with grief, symptom burden, income, disease stage, and utilization of counseling services. Caregiver ratings of patient general QOL were associated with patient symptom burden, patient grief, patient global function, caregiver burden, and caregiver spiritual wellbeing.There are notable differences in the predictors of general QOL, health-related QOL and caregiver perspectives on patient general QOL. These differences have important implications for clinical research and models of clinical care.

Background and aims: Alteration in intestinal permeability is the main factor underlying the pathogenesis of many diseases affecting the gut, such as inflammatory bowel disease [IBD]. Characterization of molecules targeting the restoration of intestinal barrier integrity is therefore vital for the development of alternative therapies. The yeast Saccharomyces boulardii CNCM I-745 [Sb], used to prevent and treat antibiotic-associated infectious and functional diarrhea, may have a beneficial effect in the treatment of IBD.Methods: We analyzed the impact of Sb supernatant on tissue integrity and components of adherens junctions using cultured explants of colon from both IBD and healthy patients. To evaluate the pathways by which Sb regulates the expression of E-cadherin at the cell surface, we developed in vitro assays using human colonic cell lines, including cell aggregation, a calcium switch assay, real-time measurement of transepithelial electrical resistance [TEER] and pulse-chase experiments.Results: We showed that Sb supernatant treatment of colonic explants protects the epithelial morphology and maintains E-cadherin expression at the cell surface. In vitro experiments revealed that Sb supernatant enhances E-cadherin delivery to the cell surface by re-routing endocytosed E-cadherin back to the plasma membrane. This process, involving Rab11A-dependent recycling endosome, leads to restoration of enterocyte adherens junctions, in addition to the overall restoration and strengthening of intestinal barrier function.Conclusion: These findings open new possibilities of discovering novel options for prevention and therapy of diseases that affect intestinal permeability.

Malignant transformation of melanocytes frequently coincides with an alteration in the expression of cell–cell adhesion molecules (cadherins) and cell-extracellular matrix proteins (integrins). How these two adhesion systems interplay to impact on cell invasion remains to be described in melanoma. Cell adhesion networks were localised by immunofluorescence in human primary cutaneous melanoma, metastatic melanoma in the lymph nodes, and melanoma cell lines. The role of these cell adhesion networks was assessed both in vivo, by analysing their impact on tumour growth in mice, and in vitro, with the use of functional tests including cell aggregation and cell migration. We found that α2β1 integrin associates with both E-cadherin and N-cadherin to form two adhesive networks, distinguishable by the interaction—or not—of α2β1 integrin with type I collagen. N-cadherin/α2β1 integrin and E-cadherin/α2β1 integrin networks differently participated towards tumour growth in mice. The N-cadherin/α2β1 integrin network showed specific involvement in melanoma cell invasion and migration towards type I collagen. On the other hand, the E-cadherin/α2β1 network regulated cell–cell adhesion. This suggests that different signalling environments can be generated, depending on the type and/or local concentration of cadherin present in the adhesion complex, which potentially leads to differential cell responses. Further clarification of how these adhesive networks are regulated is fundamental to understanding important physiological and pathological processes such as morphogenesis, wound healing, tumour invasion and metastasis.

Salmonella enterica serovar Typhimurium (ST) is an enteropathogenic Gram-negative bacterium that causes infection following oral ingestion. ST spreads rapidly along the gastrointestinal tract (GIT) and invades the intestinal epithelium to ultimately reach internal body organs. The probiotic yeast Saccharomyces boulardii BIOCODEX (S.b-B) is prescribed for prophylaxis of diarrheal infectious diseases. We previously showed that S.b-B prevents weight loss in ST-infected mice and significantly decreases bacterial translocation to the spleen and liver. This study was designed to investigate the effect of S.b-B on ST migration along the GIT and the impact of the yeast on the host's early innate immune responses. Bioluminescent imaging (BLI) was used to evaluate the effect of S.b-B on the progression of luminescent Salmonella Typhimurium (ST-lux) in the GIT of mice pretreated with streptomycin. Photonic emission (PE) was measured in GIT extracts (stomach, small intestine, cecum and colon) at various time periods post-infection (PI). PE analysis revealed that, 45 min PI, ST-lux had migrated slightly faster in the mice treated with S.b-B than in the untreated infected animals. At 90 min PI, ST-lux had reached the cecum in both groups of mice. Adhesion of ST to S.b-B was visualized in the intestines of the mice and probably accounts for (1) the faster elimination of ST-lux in the feces, and (2) reduced translocation of ST to the spleen and liver. In the early phase of infection, S.b-B also modifies the host's immune responses by (1) increasing IFN-γ gene expression and decreasing IL-10 gene expression in the small intestine, and (2) elevating both IFN-γ, and IL-10 mRNA levels in the cecum. BLI revealed that S.b-B modifies ST migration and the host immune response along the GIT. Study findings shed new light on the protective mechanisms of S.b-B during the early phase of Salmonella pathogenesis.

Intestinal epithelial cell damage is frequently seen in the mucosal lesions of infectious or inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Complete remission of these diseases requires both the disappearance of inflammation and the repair of damaged epithelium. Saccharomyces boulardii (Sb, Biocodex) is a non-pathogenic yeast widely used as a preventive and therapeutic probiotic for the prevention and treatment of diarrhea and other gastrointestinal disorders. We recently showed that it enhances the repair of intestinal epithelium through activation of α2β1 integrin collagen receptors. In the present study, we demonstrated that α2β1 integrin is not the sole cell-extracellular matrix receptor involved during Sb-mediated intestinal restitution. Indeed, by using cell adhesion assays, we showed that Sb supernatant contains heat sensitive molecule(s), with a molecular weight higher than 9 kDa, which decreased αvβ5 integrin-mediated adhesion to vitronectin by competing with the integrin. Moreover, Sb-mediated changes in cell adhesion to vitronectin resulted in a reduction of the αvβ5signaling pathway. We used a monolayer wounding assay that mimics in vivo cell restitution to demonstrate that down-modulation of the αvβ5 integrin-vitronectin interaction is related to Sb-induced cell migration. We therefore postulated that Sb supernatant contains motogenic factors that enhance cell restitution through multiple pathways, including the dynamic fine regulation of αvβ5 integrin binding activity. This could be of major importance in diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.

Intestinal epithelial cell damage is frequently seen in the mucosal lesions of inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Complete remission of these diseases requires both the cessation of inflammation and the migration of enterocytes to repair the damaged epithelium. Lyophilized Saccharomyces boulardii (Sb, Biocodex) is a nonpathogenic yeast widely used as a therapeutic agent for the treatment and prevention of diarrhea and other gastrointestinal disorders. In this study, we determined whether Sb could accelerate enterocyte migration. Cell migration was determined in Sb force-fed C57BL6J mice and in an in vitro wound model. The impact on α2β1 integrin activity was assessed using adhesion assays and the analysis of α2β1 mediated signaling pathways both in vitro and in vivo. We demonstrated that Sb secretes compounds that enhance the migration of enterocytes independently of cell proliferation. This enhanced migration was associated with the ability of Sb to favor cell-extracellular matrix interaction. Indeed, the yeast activates α2β1 integrin collagen receptors. This leads to an increase in tyrosine phosphorylation of cytoplasmic molecules, including focal adhesion kinase and paxillin, involved in the integrin signaling pathway. These changes are associated with the reorganization of focal adhesion structures. In conclusion Sb secretes motogenic factors that enhance cell restitution through the dynamic regulation of α2β1 integrin activity. This could be of major importance in the development of novel therapies targeting diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.

BACKGROUND INFORMATION:Previous studies have reported that cross-talk between integrins may be an important regulator of integrin-ligand binding and subsequent signalling events that control a variety of cell functions in many tissues. We previously demonstrated that αvβ5/β6 integrin represses α2β1-dependent cell migration. The αv subunits undergo an endoproteolytic cleavage by protein convertases, whose role in tumoral invasion has remained controversial.RESULTS:Inhibition of convertases by the convertase inhibitor α1-PDX (α1-antitrypsin Portland variant), leading to the cell-surface expression of an uncleaved form of the αv integrin, stimulated cell migration toward type I collagen. Under convertase inhibition, α2β1 engagement led to enhanced phosphorylation of both FAK (focal adhesion kinase) and MAPK (mitogen-activated protein kinase). This outside-in signalling stimulation was associated with increased levels of activated β1 integrin located in larger than usual focal-adhesion structures and a cell migration that was independent of the PI3K (phosphoinositide 3-kinase)/Akt (also called protein kinase B) pathway.CONCLUSIONS:The increase in cell migration observed upon convertases inhibition appears to be due to the up-regulation of β1 integrins and to their location in larger focal-adhesion structures. The endoproteolytic cleavage of αv subunits is necessary for αvβ5/β6 integrin to control α2β1 function and could thus play an essential role in colon cancer cell migration.

Cell adhesion molecules, including cadherins and integrins, play an essential role during tumor progression and represent potential targets for the development of new therapeutic agents. We previously showed that lebectin, a C-type lectin protein (CLP) issued from Macrovipera lebectina snake venom, inhibits integrin-mediated migration of IGR39 melanoma cells. Here we assessed whether lebectin modulates cell-cell adhesion. We demonstrated that lebectin promotes N-cadherin/catenin complex reorganization at cell-cell contacts, inducing a strengthening of intercellular adhesion. This reorganization is associated to phosphorylation of beta-catenin on tyrosine 142 residue. Interestingly, lebectin acts on N-cadherin-mediated cell-cell contacts through PI3K/Akt pathway. This effect could contribute to the blockage of tumor cell migration previously observed.

L'épithélium intestinal est constamment exposé à des agents nocifs endogènes ou exogènes capables d'entraîner des lésions. L'intégrité de l'épithélium est rapidement rétablie du fait de la capacité de la muqueuse intestinale à se régénérer via un processus migratoire appelé restitution intestinale. Toutefois, dans certaines pathologies inflammatoires ou infectieuses, la réparation est perturbée. Saccharomyces boulardii (S. Boulardii) est une souche de levures non pathogène. Cette souche est utilisée en tant que probiotique dans la prévention de la diarrhée associée aux antibiotiques et dans la prise en charge des gastroentérites. Des observations réalisées in vivo suggèrent que S. Boulardii pourrait favoriser la restitution intestinale. Le but de notre étude a été d'observer l'effet du surnageant de S. Boulardii sur la restauration de la muqueuse intestinale in vitro. Nous avons cherché, au moyen d'un test de blessure recolonisation, si le surnageant de S. Boulardii modulait le pouvoir migratoire de quatre lignées coliques humaines (HCT-8/E11, HT29-D4, CaCo2/TC7 et T84). Nous montrons que (1) S. boulardii est capable d'accélérer la réparation de l'épithélium intestinal en accélérant la migration cellulaire. (2) Cet effet ne s'exerce que sur des cellules épithéliales différenciées. (3) La migration est indépendante de la prolifération cellulaire. (4) S. boulardii module l'activité des intégrines α2β1 et αvβ5, récepteurs respectivement du collagène de type I et de la vitronectine. C'est donc en modulant les interactions intégrines-matrice extracellulaire que S. Boulardii pourrait exercer un pouvoir pro-migratoire. A notre connaissance, c'est la première démonstration d'un effet promigratoire de S. Boulardii sur les cellules épithéliales intestinales. Les propriétés motogènes de S. Boulardii sur le processus de migration cellulaire, et donc de régénération de l'épithélium intestinal, pourraient être mises à profit pour le traitement de certaines maladies inflammatoires et infectieuses dans lesquelles la réparation intestinale est fortement perturbée.

Crosstalk between integrins is involved in the regulation of various cell functions including cell migration. Here we identify the interplay between the integrins alphavbeta5/beta6 and alpha2beta1 during cell migration toward type I collagen. Human colon cancer cell lines HT29-D4 and SW480 were used as cell models. To improve our understanding of the consequences of alphavbeta5/beta6 function on alpha2beta1, we decreased the expression of alphav integrins by either siRNA or lysosomal targeting strategies or inhibited their function using, as antagonists, blocking antibodies or disintegrins. In all cases, we observed a greatly enhanced alpha2beta1 integrin-dependent cell migration associated with focal adhesion rearrangements and increased outside-in signaling as demonstrated by elevated phosphorylation of focal adhesion kinase and MAPKinase (ERK1 and ERK2). The alphavbeta5/beta6-dependent limitation of alpha2beta1 function could be overridden by TS2/16, an activating anti-beta1 antibody. Interestingly, compared to control cells, the pharmacological inhibition of PI3Kinase or the siRNA-mediated knockdown of AKT had little effect on the high alpha2beta1-mediated cell migration observed in the absence of alphav integrins or following activation of alpha2beta1 integrins by the TS2/16. These results suggest that integrins alphavbeta5/beta6 repress alpha2beta1 possibly by interfering with their activation process and thereby modify the cell signaling regulation of alpha2beta1-mediated migration.

Abstract The poor prognosis of pancreatic cancer is due to rapid locoregional invasion, the early development of metastases, and the limited efficacy of current therapies. To date, none of the identified oncogenes and suppressors involved in this disease have led to efficient treatments. Here, we describe that the scaffold protein ArgBP2 is repressed during oncogenic transformation of the pancreas. We could show, using a pancreatic cancer cell line model, that this repression of ArgBP2 participates in the progression of this disease. Interestingly, in vitro analyses revealed that the antitumoral potential of ArgBP2 is linked to the control of cell adhesion and migration rather than to the regulation of cell proliferation or sensitivity to apoptosis. Moreover, we could detail part of the molecular mechanism responsible by identifying new ArgBP2-interacting proteins, and show that this function is partly achieved by the control of a WAVE/PTP-PEST/c-Abl signaling complex. These findings point to a new mechanism of pancreatic cancer progression leading to invasion and metastasis and suggest that the ArgBP2 signaling pathway could represent a new target for cancer therapy. [Cancer Res 2008;68(12):4588–96]