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Epithelial to mesenchymal transition (EMT) is a key step toward metastasis. MCF7 breast cancer cells conditionally expressing the EMT master regulator SNAI1 were used to identify early expressed microRNAs (miRNAs) and their targets that may contribute to the EMT process. Potential targets of miRNAs were identified by matching lists of in silico predicted targets and of inversely expressed mRNAs. MiRNAs were ranked based on the number of predicted hits, highlighting miR-661, a miRNA with so far no reported role in EMT. MiR-661 was found required for efficient invasion of breast cancer cells by destabilizing two of its predicted mRNA targets, the cell-cell adhesion protein Nectin-1 and the lipid transferase StarD10, resulting, in turn, in the downregulation of epithelial markers. Reexpression of Nectin-1 or StarD10 lacking the 3'-untranslated region counteracted SNAI1-induced invasion. Importantly, analysis of public transcriptomic data from a cohort of 295 well-characterized breast tumor specimen revealed that expression of StarD10 is highly associated with markers of luminal subtypes whereas its loss negatively correlated with the EMT-related, basal-like subtype. Collectively, our non-a priori approach revealed a nonpredicted link between SNAI1-triggered EMT and the down-regulation of Nectin-1 and StarD10 through the up-regulation of miR-661, which may contribute to the invasion of breast cancer cells and poor disease outcome.

Background: The majority of human cancer deaths are caused by metastasis. The metastatic dissemination is initiated by the breakdown of epithelial cell homeostasis. During this phenomenon, referred to as epithelial to mesenchymal transition (EMT), cells change their genetic and trancriptomic program leading to phenotypic and functional alterations. The challenge of understanding this dynamic process resides in unraveling regulatory networks involving master transcription factors (e.g. SNAI1/2, ZEB1/2 and TWIST1) and microRNAs. Here we investigated microRNAs regulated by SNAI1 and their potential role in the regulatory networks underlying epithelial plasticity. Results: By a large-scale analysis on epithelial plasticity, we highlighted miR-203 and its molecular link with SNAI1 and the miR-200 family, key regulators of epithelial homeostasis. During SNAI1-induced EMT in MCF7 breast cancer cells, miR-203 and miR-200 family members were repressed in a timely correlated manner. Importantly, miR-203 repressed endogenous SNAI1, forming a double negative miR203/SNAI1 feedback loop. We integrated this novel miR203/SNAI1 with the known miR200/ZEB feedback loops to construct an a priori EMT core network. Dynamic simulations revealed stable epithelial and mesenchymal states, and underscored the crucial role of the miR203/SNAI1 feedback loop in state transitions underlying epithelial plasticity. Conclusion: By combining computational biology and experimental approaches, we propose a novel EMT core network integrating two fundamental negative feedback loops, miR203/SNAI1 and miR200/ZEB. Altogether our analysis implies that this novel EMT core network could function as a switch controlling epithelial cell plasticity during differentiation and cancer progression.

BACKGROUND: Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion. METHODOLOGY/PRINCIPAL FINDINGS: To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-delta isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process. CONCLUSIONS/SIGNIFICANCE: Altogether these findings quantitatively demon

The transcription regulator SNAI1 triggers a transcriptional program leading to epithelial to mesenchymal transition (EMT), providing epithelial cells with mesenchymal features and invasive properties during embryonic development and tumor progression. To identify early transcriptional changes occurring during SNAI1-induced EMT, we performed a time-resolved genome-scale study using human breast carcinoma cells conditionally expressing SNAI1. The approach we developed for microarray data analysis, allowed identifying three distinct EMT stages and the temporal classification of genes. Importantly, we identified unexpected, biphasic expression profiles of EMT-associated genes, supporting their pivotal role during this process. Finally, we established early EMT gene networks by identifying transcription factors and their potential targets which may orchestrate early events of EMT. Collectively, our work provides a framework for the identification and future systematic analysis of novel genes which contribute to SNAI1-triggered EMT.

Talin establishes a major link between integrins and actin filaments and contains two distinct integrin binding sites: one, IBS1, located in the talin head domain and involved in integrin activation and a second, IBS2, that maps to helix 50 of the talin rod domain and is essential for linking integrin beta subunits to the cytoskeleton ( Moes, M., Rodius, S., Coleman, S. J., Monkley, S. J., Goormaghtigh, E., Tremuth, L., Kox, C., van der Holst, P. P., Critchley, D. R., and Kieffer, N. (2007) J. Biol. Chem. 282, 17280-17288 ). Through the combined approach of mutational analysis of the beta3 integrin cytoplasmic tail and the talin rod IBS2 site, SPR binding studies, as well as site-specific antibody inhibition experiments, we provide evidence that the integrin beta3-talin rod interaction relies on a helix-helix association between alpha-helix 50 of the talin rod domain and the membrane-proximal alpha-helix of the beta3 integrin cytoplasmic tail. Moreover, charge complementarity between the highly conserved talin rod IBS2 lysine residues and integrin beta3 glutamic acid residues is necessary for this interaction. Our results support a model in which talin IBS2 binds to the same face of the beta3 subunit cytoplasmic helix as the integrin alphaIIb cytoplasmic tail helix, suggesting that IBS2 can only interact with the beta3 subunit following integrin activation.

Talin is a multifunctional cytoskeletal protein that plays a critical role in linking the actin cytoskeleton to the integrin family of transmembrane cell adhesion receptors. Two distinct integrin binding sites have been identified in talin, one present in the globular head domain (IBS1) and involved in integrin activation, and a second (IBS2), that has been delineated to a 130 residue fragment of the talin rod domain, but whose functional role is still elusive (Tremuth et al.2004). The objective of the present study was to define the minimal structure of talin IBS2 and to investigate its functional role in the integrin-cytoskeleton connection.In the first part of this study, we used a combination of three different experimental approaches to define the minimal structure of talin IBS2: 1) an in silico bioinformatics approach to analyse sequence conservation of talin IBS2, 2) an in vivo cell biology approach to study the subcellular localization of recombinant talin fragments covering IBS2 in CHOáIIbâ3 cells, and 3) an in vitro biochemical approach consisting in protein overlay, pull down and Surface Plasmon Resonance (SPR) assays, to study the direct interaction between talin IBS2 and the integrin â3 subunit. We delineated IBS2 to a single amphipathic á-helical repeat of 23 residues within the talin rod domain. We further provided evidence that a two amino acid mutation(L2094I2095/AA) was sufficient to inactivate the IBS2 site, due to a disruption of the á helix structure, as demonstrated by infrared spectroscopy. In addition, we identified 2 lysine residues (K2085, K2089) exposed on the solvent face of á helix 50, which are directly involved in the talin IBS2-integrin interaction.In the second part of this study, we investigated the functional role of talin IBS2 in spreading defective talin (-/-) cells and showed that in contrast to full-length wild type talin, an IBS2 LI/AA mutant talin was unable to fully rescue the spread phenotype of these cells. These res, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Talin1 is a large cytoskeletal protein that links integrins to actin filaments through two distinct integrin binding sites, one present in the talin head domain (IBS1) necessary for integrin activation and a second (IBS2) that we have previously mapped to talin residues 1984-2113 (fragment J) of the talin rod domain (1 Tremuth, L. Kreis, S. Melchior, C. Hoebeke, J. Ronde, P. Plancon, S. Takeda, K. and Kieffer, N. (2004) J. Biol. Chem. 279, 22258-22266), but whose functional role is still elusive. Using a bioinformatics and cell biology approach, we have determined the minimal structure of IBS2 and show that this integrin binding site corresponds to 23 residues located in alpha helix 50 of the talin rod domain (residues 2077-2099). Alanine mutation of 2 highly conserved residues (L2094A/I2095A) within this alpha helix, which disrupted the alpha-helical structure of IBS2 as demonstrated by infrared spectroscopy and limited trypsin proteolysis, was sufficient to prevent in vivo talin fragment J targeting to alphaIIbbeta3 integrin in focal adhesions and to inhibit in vitro this association as shown by an alphaIIbbeta3 pulldown assay. Moreover, expression of a full-length mouse green fluorescent protein-talin LI/AA mutant in mouse talin1(-/-) cells was unable to rescue the inability of these cells to assemble focal adhesions (in contrast to green fluorescent protein-talin wild type) despite the presence of IBS1. Our data provide the first direct evidence that IBS2 in the talin rod is essential to link integrins to the cytoskeleton., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published