Aurélie Dobric PhD in cell biology

Invasion/metastasis axis is the major driver of cancer mortality. By maintaining tissue cohesion, E-cadherin is considered as a protective marker for tumoral progression. However, recent studies suggested that the appearance of hybrid epithelial-mesenchymal (E/M) cells still expressing E-cadherin is favourable for the establishment of metastasis. This hypothesis is consistent with the observation that most pancreatic ductal adenocarcinomas (PDAC) are invasive and express E-cadherin in primary tumour and metastases. By using a data constituted by a series of patient-derived xenografts (PDX) from the PaCaOmics multi-centric clinical trial, we show that E-cadherin expression is not associated with stages of the pathology, prognosis, and overall survival in PDAC. The role of E-cadherin in PDAC aggressiveness was tested both in vitro and in cancer cell implantation models. We show that E-cadherin is a key component of membrane protrusions implicated in the extracellular matrix remodelling and degradation, called invadopodia. Ecadherin downregulation in a pancreatic model of E/M hybrid cells reveals that E-cadherin downregulates Arp2/3 complex expression. As this complex is essential for branched actin structure, E-cadherin depletion strongly impaired invadopodia formation. On the other hand, we demonstrate that E-cadherin interacts with the membrane protease MT1-MMP at the invadopodial membrane. E-cadherin could be recycled back to the invadopodial membrane simultaneously with MT1-MMP.

Indeed, both Rab7 vesicle-dependant and/or a Rab11 vesicledependant pathway are required for both E-cadherin and MT1-MMP trafficking. This new localization of E-cadherin and its implication in cell invasion shines a new light on hybrid EMT features in tumoral invasion.

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

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.

The microRNA-200 (miR-200) family is frequently down-regulated in tumors, including pancreatic adenocarcinomas (PDACs). In this study we have examined the mechanisms involved in the loss of miR-200s in tumoral pancreatic cells. Whereas miR-200 gene promoters appear methylated in mature miR-200 deficient cell lines, miR-200 precursors are detected in nuclear but not cytoplasmic compartment of these cells, indicating that promoter hypermethylation is not sufficient to explain the deficit of mature miR-200s. The ribonuclease Monocyte Chemotactic Protein-induced Protein-1 (MCPiP1) may counteract Dicer1 in miRNA maturation process. MCPiP1/Dicer1 mRNA and protein ratios appear higher in miR-200 deficient compared to miR-200 proficient cells, suggesting that MCPiP1 may compete with Dicer1 in mature miR-200 deficient cells. Inhibition of MCPiP1 allows the detection of miR-200 precursors in cytoplasm of miR-200 deficient cells, confirming its involvement in the loss of miR-200s. Also, reversion of MCPiP1/Dicer1 ratio by over-expression of Dicer1 in miR-200 deficient cells leads to the recovery of mature miR-200s. Finally, whereas human malignant pancreatic tissues (PDACs) express lower miR-200 levels than non malignant tissues (non-MPDs), MCPiP1/Dicer1 ratio appears higher in PDACs, when compared to non-MPDs, supporting the hypothesis that MCPiP1/Dicer1 ratio is determinant in regulating miR-200 maturation process in a subset of tumoral pancreatic cells.

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.

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.

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 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.

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.