E47 and Id1 interplay in epithelial-mesenchymal transition.

E12/E47 proteins (encoded by E2A gene) are members of the class I basic helix-loop-helix (bHLH) transcription factors (also known as E proteins). E47 has been described as repressor of E-cadherin and inducer of epithelial-mesenchymal transition (EMT). We reported previously that EMT mediated by E47 in MDCK cells occurs with a concomitant overexpression of Id1 and Id3 proteins. Id proteins belong to class V of HLH factors that lack the basic domain; they dimerise with E proteins and prevent their DNA interaction, thus, acting as dominant negative of E proteins. Here, we show that E47 interacts with Id1 in E47 overexpressing MDCK cells that underwent a full EMT as well as in mesenchymal breast carcinoma and melanoma cell lines. By conducting chromatin immunoprecipitation assays we demonstrate that E47 binds directly to the endogenous E-cadherin promoter of mesenchymal MDCK-E47 cells in a complex devoid of Id1. Importantly, our data suggest that both E47 and Id1 are required to maintain the mesenchymal phenotype of MDCK-E47 cells. These data support the collaboration between E47 and Id1 in the maintenance of EMT by mechanisms independent of the dominant negative action of Id1 on E47 binding to E-cadherin promoter. Finally, the analysis of several N0 breast tumour series indicates that the expression of E47 and ID1 is significantly associated with the basal-like phenotype supporting the biological significance of the present findings.

Hyaluronic acid/chitosan-g-poly(ethylene glycol) nanoparticles for gene therapy: an application for pDNA and siRNA delivery.

PURPOSE: To design hyaluronic acid (HA) and chitosan-g-poly(ethylene glycol) (CS-g-PEG) nanoparticles intended for a broad range of gene delivery applications. METHODS: Nanoparticles formulated at different HA/CS-g-PEG mass ratios were developed to associate either pDNA or siRNA. The physico-chemical characteristics, morphology, association efficiency and nuclease protection ability of the nanocarriers were compared for these two molecules. Their biological performance, including transfection effciency, nanoparticle cellular uptake and citotoxicity, was assesed. RESULTS: The resulting nanoparticles showed an adequate size (between 130 and 180 nm), and their surface charge could be modulated according to the nanoparticle composition (from +30 mV to -20 mV). All prototypes exhibited a greater association efficiency and nuclease protection for pDNA than for siRNA. However, cell culture experiments evidenced that HA/CS-g-PEG nanoparticles were effective carriers for the delivery of both, siRNA and pDNA, eliciting a biological response with minimal cytotoxicity. Moreover, experiments performed in the HEK-EGFP-Snail1 cell line showed the potential of the HA/CS-g-PEG nanoparticles to silence the expression of the Snail1 transcription factor, an important mediator in tumor progression. CONCLUSIONS: HA/CS-g-PEG nanoparticles can be easily modulated for the delivery of different types of gene molecules, offering great potential for gene therapy applications, as evidenced by their biological performance.

The morphological and molecular features of the epithelial-to-mesenchymal transition.

Here we describe several methods for the characterization of epithelial-mesenchymal transition (EMT) at the cellular, molecular and behavioral level. This protocol describes both in vitro and in vivo approaches designed to analyze different features that when taken together permit the characterization of cells undergoing transient or stable EMT. We define straightforward methods for phenotypical, cellular and transcriptional characterization of EMT in vitro in monolayer cultures. The procedure also presents technical details for the generation of in vitro three-dimensional (3D) cultures analyzing cell phenotype and behavior during the EMT process. In addition, we describe xenotransplantation techniques to graft 3D cell cultures into mice to study in vivo invasion in a physiological-like environment. Finally, the protocol describes the analysis of selected EMT markers from experimental and human tumor samples. This series of methods can be applied to the study of EMT under various experimental and biological situations. Once the methodology is established, the time required to complete the protocol may vary from 3 to 4 weeks (monolayer cultures) and up to 6-8 weeks if including 3D cultures.

The class I bHLH factors E2-2A and E2-2B regulate EMT.

Functional loss of the cell-cell adhesion molecule E-cadherin is an essential event for epithelial-mesenchymal transition (EMT), a process that allows cell migration during embryonic development and tumour invasion. In most carcinomas, transcriptional repression has emerged as the main mechanism responsible for E-cadherin downregulation. Here, we report the identification of class I bHLH factor E2-2 (TCF4/ITF2) as a new EMT regulator. Both isoforms of E2-2 (E2-2A and E2-2B) induce a full EMT when overexpressed in MDCK cells but without affecting the tumorigenic properties of parental cells, in contrast to other EMT inducers, such as Snail1 or class I bHLH E47. E-cadherin repression mediated by E2-2 is indirect and independent of proximal E-boxes of the promoter. Knockdown studies indicate that E2-2 expression is dispensable for maintenance of the EMT driven by Snail1 and E47. Comparative gene-profiling analysis reveals that E2-2 factors induce similar, yet distinct, genetic programs to that induced by E47 in MDCK cells. These results, together with the embryonic expression pattern of Tcf4 and E2A (which encodes E12/E47), support a distinct role for E2-2 and suggest an interesting interplay between E-cadherin repressors in the regulation of physiological and pathological EMT processes.

Id-1 is induced in MDCK epithelial cells by activated Erk/MAPK pathway in response to expression of the Snail and E47 transcription factors.

Id-1, a member of the helix-loop-helix transcription factor family has been shown to be involved in cell proliferation, angiogenesis and invasion of many types of human cancers. We have previously shown that stable expression of E47 and Snail repressors of the E-cadherin promoter in MDCK epithelial cell line triggers epithelial mesenchymal transition (EMT) concomitantly with changes in gene expression. We show here that both factors activate the Id-1 gene promoter and induce Id-1 mRNA and protein. The upregulation of the Id-1 gene occurs through the transactivation of the promoter by the Erk/MAPK signaling pathway. Moreover, oncogenic Ras is also able to activate Id-1 promoter in MDCK cells in the absence of both E47 and Snail transcription factors. Several transcriptionally active regulatory elements have been identified in the proximal promoter, including AP-1, Sp1 and four putative E-boxes. By EMSA, we only detected an increased binding to Sp1 and AP-1 elements in E47- and Snail-expressing cells. Binding is affected by the treatment of cells with PD 98059 MEK inhibitor, suggesting that MAPK/Erk contributes to the recruitment or assembly of proteins to Id-1 promoter. Small interfering RNA directed against Sp1 reduced Id-1 expression and the upregulation of the promoter, indicating that Sp1 is required for Id-1 induction in E47- and Snail-expressing cells. Our results provide new insights into how some target genes are activated during and/or as a consequence of the EMT triggered by both E47 and Snail transcription factors.

Genetic profiling of epithelial cells expressing E-cadherin repressors reveals a distinct role for Snail, Slug, and E47 factors in epithelial-mesenchymal transition.

The transcription factors Snail, Slug, and bHLH E47 have been recently described as direct repressors of E-cadherin and inducers of epithelial-mesenchymal transition (EMT) and invasion when overexpressed in epithelial cells. Although a role of those factors in tumor progression and invasion has been proposed, whether the different repressors play distinct or redundant roles in the tumorigenic process has not been established. To further investigate this important issue, we have analyzed the gene expression profiling of Madin-Darby canine kidney (MDCK) epithelial cells expressing the different repressors (MDCK-Snail, MDCK-Slug, and MDCK-E47 cells) versus control MDCK cells by cDNA microarrays. A total of 243 clones (228 genes and 15 expressed sequence tags) were found to be differentially expressed between either of the three MDCK-derived cell lines and control MDCK cells. Twenty two of the candidate genes were validated by Northern blot, Western blot, immunofluorescence, and promoter analyses in cell lines and by immunohistochemistry in xenografted tumors. Gene clustering analysis indicated that about a third of the 243 candidate genes were common to MDCK cells expressing Snail, Slug, or E47 factors, whereas the rest of the genes were regulated in only one or two cell types. Differentially regulated genes include those related to EMT (45 genes), transcriptional regulation (18 genes), cell proliferation and signaling (54 genes), apoptosis (12 genes), and angiogenesis (9 genes). These results indicate that Snail, Slug, and E47 transcription factors induce common and specific genetic programs, supporting a differential role of the factors in tumor progression and invasion.

Upregulation of MMP-9 in MDCK epithelial cell line in response to expression of the Snail transcription factor.

Overexpression of the transcription factor Snail in epithelial MDCK cells promotes the epithelial-mesenchymal transition (EMT) and the acquisition of an invasive phenotype. We report here that the expression of Snail is associated with an increase in the promoter activity and expression of the matrix metalloproteinase MMP-9. The effect of Snail silencing on MMP-9 expression corroborates this finding. Induced transcription of MMP-9 by Snail is driven by a mechanism dependent on the MAPK and phosphoinositide 3-kinase (PI3K) signalling pathways. Although other regions of the promoter were required for a complete stimulation by Snail, a minimal fragment (nucleotides -97 to +114) produces a response following an increased phosphorylation of Sp-1 and either Sp-1 or Ets-1 binding to the GC-box elements contained in this region. The expression of a dominant negative form of MEK decreased these complexes. A moderate increase in the binding of the nuclear factor kappaB (NFkappaB) to the upstream region (nucleotide -562) of the MMP-9 promoter was also observed in Snail-expressing cells. Interestingly, oncogenic H-Ras (RasV12) synergistically co-operates with Snail in the induction of MMP-9 transcription and expression. Altogether, these results indicate that MMP-9 transcription is activated in response to Snail expression and that it might explain, at least in part, the invasive properties of the Snail-expressing cells.

Snail and E47 repressors of E-cadherin induce distinct invasive and angiogenic properties in vivo.

The transcription factors Snail and E47 are direct repressors of E-cadherin, with both inducing a full epithelial-mesenchymal transition and invasive behaviour in vitro when expressed in the prototypic epithelial MDCK cell line. The role of these repressors in the invasive process and in other tumorigenic properties is, nevertheless, still poorly understood. However, organotypic cultures and in vivo transplantation assays indicate that cells expressing MDCK-Snail and MDCK-E47 exhibit significant differences. MDCK-Snail cells have a higher infiltrative potential than MDCK-E47 cells. Interestingly, both cell types induce angiogenesis of the host stromal tissue in transplantation assays, but this property is greatly enhanced in transplants of MDCK-E47 cells. Xenografted tumours induced in nude mice also show signs of strong angiogenic potential, again markedly increased in tumours induced by MDCK-E47 which exhibit a higher vessel density and proliferation rate than those induced by MDCK-Snail cells. These results suggest differential roles for Snail and E47 E-cadherin repressors in tumour progression where Snail is implicated in promoting the initial invasion and E47 plays an active role in tumour cell growth by promoting angiogenesis.