Hélène Feracci

Cadherin-Cadherin Engagement Promotes Cell Survival via Rac1/Cdc42 and Signal Transducer and Activator of Transcription-3

Signal transducer and activator of transcription-3 (Stat3) is activated by a number of receptor and nonreceptor tyrosine kinases, whereas a constitutively active form of Stat3 alone is sufficient to induce neoplastic transformation. In the present report, we show that Stat3 can also be activated through homophilic interactions by the epithelial (E)-cadherin. Indeed, by plating cells onto surfaces coated with fragments encompassing the two outermost domains of this cadherin, we clearly show that cadherin engagement can activate Stat3, even in the absence of direct cell-to-cell contact. Most importantly, our results also reveal for the first time an unexpected and dramatic surge in total Rac1 and Cdc42 protein levels triggered by cadherin engagement and an increase in Rac1 and Cdc42 activity, which is responsible for the Stat3 stimulation observed. Inhibition of cadherin interactions using a peptide, a soluble cadherin fragment, or genetic ablation induced apoptosis, points to a significant role of this pathway in cell survival signaling, a finding that could also have important therapeutic implications. (Mol Cancer Res 2009;7(8):1310–27)

Creating Biomimetic Surfaces through Covalent and Oriented Binding of Proteins

This manuscript describes a novel method for the biofunctionalization of glass surfaces with polyhistidine-tagged proteins. The main innovation of this methodology consists of the covalent binding between the nitrilotriacetic acid (NTA) moiety and the proteins, ensuring not only orientation, but also stability of the recombinant proteins on NTA-covered surfaces. In this work, as C-terminal polyhistidine tagged cadherin extracellular fragments have been used, this methodology guarantees the proper orientation of these proteins, by mimicking their insertion into cell plasma membranes. These biofunctionalized surfaces have been characterized by confocal microscopy, X-ray photoelectron spectroscopy, contact angle, and atomic force microscopy, showing a high density of cadherins on the glass surfaces and the stability of the linkage. The prepared materials exhibited a high tendency to promote cell spreading, demonstrating the functionality of the protein and the high utility of these biomaterials to promote cell adhesion events. Interestingly, differences in the cytoskeleton organization have been observed in cells adhering to surfaces with no cadherins or with nonoriented cadherins, in comparison to surfaces functionalized with well-oriented cadherins. This method, which allows the robust immobilization of polyhistidine tagged proteins due to their covalent binding and with a defined orientation, may also find particular usefulness in the making of protein biochips, for analysis of protein-protein interactions, as well as structural and single-molecule studies.

Activated Rac1 requires gp130 for Stat3 activation, cell proliferation and migration.

Rac1 (Rac) is a member of the Rho family of small GTPases which controls cell migration by regulating the organization of actin filaments. Previous results suggested that mutationally activated forms of the Rho GTPases can activate the Signal Transducer and Activator of Transcription-3 (Stat3), but the exact mechanism is a matter of controversy. We recently demonstrated that Stat3 activity of cultured cells increases dramatically following E-cadherin engagement. To better understand this pathway, we now compared Stat3 activity levels in mouse HC11 cells before and after expression of the mutationally activated Rac1 (Rac(V12)), at different cell densities. The results revealed for the first time a dramatic increase in protein levels and activity of both the endogenous Rac and Rac(V12) with cell density, which was due to inhibition of proteasomal degradation. In addition, Rac(V12)-expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that Rac(V12) is able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that Rac(V12) expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity, indicating that these cytokines may be responsible for the Stat3 activation by Rac(V12). The upregulation of IL6 family cytokines was required for cell migration and proliferation induced by Rac(V12), as shown by gp130 knockdown experiments, thus demonstrating that the gp130/Stat3 axis represents an essential effector of activated Rac for the regulation of key cellular functions.

Classical cadherins control survival through the gp130/Stat3 axis.

Stat3 (Signal Transducer and Activator of Transcription-3) is activated by a number of receptor and nonreceptor tyrosine kinases. We recently demonstrated that engagement of E-cadherin, a calcium-dependent, cell to cell adhesion molecule which is often required for cells to remain tightly associated within the epithelium, also activates Stat3. We now examined the effect of two other classical cadherins, cadherin-11 and N-cadherin, whose expression often correlates with the epithelial to mesenchymal transition occurring in metastasis of carcinoma cells, upon Stat3 phosphorylation and activity. Our results indicate that engagement of these two cadherins also, can trigger a dramatic surge in Stat3 activity. This activation occurs through upregulation of members of the IL6 family of cytokines, and it is necessary for cell survival, proliferation and migration. Interestingly, our results also demonstrate for the first time that, in sharp contrast to Stat3, the activity of Erk (Extracellular Signal Regulated kinase) was unaffected by cadherin-11 engagement. Further examination indicated that, although IL6 was able to activate Erk in sparsely growing cells, IL6 could not induce an increase in Erk activity levels in densely growing cultures. Most importantly, cadherin-11 knock-down did allow Erk activation by IL6 at high densities, indicating that it is indeed cadherin engagement that prevents Erk activation by IL6. The fact that the three classical cadherins tested so far, E-cadherin, N-cadherin and cadherin11, which are present in essentially all tissues, actually activate Stat3 regardless of their role in metastasis, argues for Stat3 as a central survival, rather than invasion factor.

Engaged for survival From cadherin ligation to STAT3 activation

In normal tissues or tumors, cells have extensive opportunities for adhesion to their neighbors. This state is mimicked by dense cell cultures. In this review, we integrate some recent findings on a key signal transducer, STAT3 (signal transducer and activator of transcription-3), whose activity is dramatically increased following cadherin-mediated cell to cell adhesion. Cadherin engagement, favored in dense cell cultures, causes a dramatic increase in total Rac/Cdc42 protein levels through inhibition of proteasomal degradation, which is followed by activation of IL-6 and STAT3. The cadherin/Rac/IL-6/STAT3 axis offers a potent survival signal that is a prerequisite for neoplastic transformation, as well as normal tissue function.

Surface engineered magnetic nanoparticles for specific immunotargeting of cadherin expressing cells

This work was supported by SAF2014-54763-C2-R (Ministerio de Economia y Competitividad), ERC-Starting Grant 239931-NANOPUZZLE project, Fondo Social Europeo (FSE; Gobierno de Aragon), Communaute de Travail des Pyrenees (P5/13 Gobierno de Aragon and 13010774 Region Aquitaine), SUDOE Train2 program and institutional funding from CNRS. BS was supported by a post-doctoral fellowship from the Instituto de Salud Carlos III in the Sara Borell program.

Chapter 8 – Organic Nanoparticles

Organic nanoparticles/nanobeads are of major interest in the material and life sciences. Biopolymer nanoparticles offer several advantages, which include the ease of their preparation from well-understood biodegradable polymers and their high stability in biological fluids and during storage. In this chapter, examples from three different fields—nucleic acid delivery, immunoassays, and cell adhesion—were chosen in order to provide a prospective of the applications of such nanomaterials to biotechnology and biomedicine.

Abstract 991: Cadherin-cadherin engagement promotes cell survival via Rac/Cdc42 and Stat3

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnStat3 (signal transducer and activator of transcription-3) is activated by a number of receptor and non-receptor tyrosine kinases, while a constitutively active form of Stat3 alone is sufficient to induce neoplastic transformation. We recently demonstrated a dramatic increase in the activity of Stat3 in breast carcinoma as well as normal epithelial cells and fibroblasts, as a consequence of cell to cell adhesion (Oncogene 23:2600). Given the generally accepted, positive role of Stat3 in proliferation, the Stat3 activity increase observed in confluent cells, that is when cells do not divide, was an unexpected observation. Interestingly, by plating cells onto surfaces coated with fragments encompassing the two outermost domains of E-cadherin and cadherin-11, two members of the classical type I and II cadherin family of surface receptors, responsible for the formation of cell to cell junctions, we demonstrated that cadherin engagement per se can directly activate Stat3, in the absence of cell to cell contact. Examination of the mechanism of the cadherin-mediated, Stat3 activation unexpectedly revealed for the first time a dramatic surge in total Rac1 and Cdc42 protein levels by cadherin engagement, and a proportional increase in Rac1 and Cdc42 activity. Therefore, to examine the potential role of Rac/Cdc42 in the density-dependent, Stat3 activation, the ability of mutationally activated RacV12 to activate Stat3 at high cell densities was examined. The results revealed a dramatic increase in protein levels and activity of both the endogenous Rac and RacV12 with cell density, which was due to inhibition of proteasomal degradation in both cases. In addition, RacV12-expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that RacV12 is, in fact, able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that both cadherin engagement and RacV12 expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity in densely growing normal, as well as in RacV12-transformed cells, indicating that the IL6 family may be responsible for the Stat3 activation both by cadherin engagement and Rac mutational activation. Indeed, Rac knockdown reduced the density-mediated, Stat3 activation, indicating that Rac is responsible for the Stat3 stimulation observed upon cadherin ligation. Inhibition of cadherin interactions using a peptide, a soluble cadherin fragment or genetic ablation induced apoptosis, pointing to a significant role of this pathway in cell survival signalling, a finding which could also have important therapeutic implications. (supported by CIHR, CBCF-Ontario chapter, US Army breast cancer program, NSERC and Breast Cancer Action Kingston).nnNote: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 991.