Structure-Based Virtual Screening Allows the Identification of Efficient Modulators of E-Cadherin-Mediated Cell-Cell Adhesion.

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.

Dendritic cell-based vaccination: powerful resources of immature dendritic cells against pancreatic adenocarcinoma.

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.

Cadherin-1 and cadherin-3 cooperation determines the aggressiveness of pancreatic ductal adenocarcinoma.

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.

Saccharomyces boulardii CNCM I-745 Restores intestinal Barrier Integrity by Regulation of E-cadherin Recycling.

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.

Interplay between cadherins and α2ß1 integrin differentially regulates melanoma cell invasion.

BACKGROUND: 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. METHODS: 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. RESULTS: We found that α2ß1 integrin associates with both E-cadherin and N-cadherin to form two adhesive networks, distinguishable by the interaction-or not-of α2ß1 integrin with type I collagen. N-cadherin/α2ß1 integrin and E-cadherin/α2ß1 integrin networks differently participated towards tumour growth in mice. The N-cadherin/α2ß1 integrin network showed specific involvement in melanoma cell invasion and migration towards type I collagen. On the other hand, the E-cadherin/α2ß1 network regulated cell-cell adhesion. CONCLUSIONS: 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.

A pancreatic tumor-specific biomarker characterized in humans and mice as an immunogenic onco-glycoprotein is efficient in dendritic cell vaccination.

Oncofetal fucose-rich glycovariants of the pathological bile salt-dependent lipase (pBSDL) appear during human pancreatic oncogenesis and are detected by themonoclonal antibody J28 (mAbJ28). We aimed to identify murine counterparts onpancreatic ductal adenocarcinoma (PDAC) cells and tissue and investigate the potential of dendritic cells (DC) loaded with this unique pancreatic tumor antigen to promote immunotherapy in preclinical trials. Pathological BSDLs purified from pancreatic juices of patients with PDAC were cleaved to generate glycosylated C-terminal moieties (C-ter) containing mAbJ28-reactive glycoepitopes. Immunoreactivity of the murine PDAC line Panc02 and tumor tissue to mAbJ28 was detected by immunohistochemistry and flow cytometry. C-ter-J28+ immunization promoted Th1-dominated immune responses. In vitro C-ter-J28+-loaded DCskewed CD3+ T-cells toward Th1 polarization. C-ter-J28+-DC-vaccinations selectively enhanced cell immunoreactivity to Panc02, as demonstrated by CD4+- and CD8+-T-cell activation, increased percentages of CD4+- and CD8+-T-cells and NK1.1+ cells expressing granzyme B, and T-cell cytotoxicity. Prophylactic and therapeutic C-ter-J28+-DC-vaccinations reduced ectopic Panc02-tumor growth, provided long-lasting protection from Panc02-tumor development in 100% of micebut not from melanoma, and attenuated progression of orthotopic tumors as revealed by MRI. Thusmurine DC loaded with pancreatic tumor-specific glycoepitope C-ter-J28+ induce efficient anticancer adaptive immunity and represent a potential adjuvant therapy for patients afflicted with PDAC.