Epithelial–mesenchymal transition

Epithelial–mesenchymal transition or transformation (EMT) is a hypothesized program of development of biological cells characterized by loss of cell adhesion, repression of E-cadherin expression, and increased cell mobility. EMT may be essential for numerous developmental processes including mesoderm formation and neural tube formation.

The concept
EMT was first recognized as a feature of embryogenesis, which is vital for morphogenesis during embryonic development. In vertebrates, epithelium and mesenchyme are the basic tissue phenotypes. The EMT can be defined as a process that produces complete loss of epithelial traits by the former epithelial cells accompanied by total acquisition of mesenchymal characteristics, such as vimentin, myosin, invasive motility, and so on. Although EMT evolved among several other animals, the remarkable ability of developing embryos to change one tissue type to the other reached its peak in the vertebrates. EMT takes place during the construction of the vertebral column out of the extracellular matrix, which is to be synthesized by fibroblasts and osteoblasts that encircle the neural tube. The major source of these cells are sclerotome and somite mesenchyme as well as primitive streak. Mesenchymal morphology allows the cells to travel to specific targets in the embryo, where they differentiate and/or induce differentiation of other cells.

In the lower chordates, gastrulation is a totally epithelial event. The amphibians form a blastopore through which presumptive mesodermal and endodermal epithelia invaginate. Amphioxus forms an epithelial neural tube and dorsal notochord but does not have the EMT potential of the primitive streak. In higher chordates, the mesenchyme originated out of the primitive streak migrates anteriorly to form the somites and participate with neural crest mesenchyme in formation of the heart mesoderm. Mesenchymal cells from the primitive streak participate also in the formation of many epithelial mesodermal organs, such as notochord as well as somites. This process involves mesenchymal–epithelial transition.

One more source of embryonic mesenchyme is the dorsal neural tube epithelium. It forms the neural crest and considerable amounts of craniofacial crest mesenchyme which forms connective tissue that contributes to the head and face in the vertebrate.

Induction
Several oncogenic pathways (peptide growth factors, Src, Ras, Ets, integrin, Wnt/beta-catenin and Notch) may induce EMT. In particular, Ras-MAPK has been shown to activate two related transcription factors known as Snail and Slug. Both of these proteins are transcriptional repressors of E-cadherin and their expression induces EMT. Interestingly, Slug triggers the steps of desmosomal disruption, cell spreading, and partial separation at cell–cell borders, which comprise the first and necessary phase of the EMT process. On the other hand, Slug cannot trigger the second phase, which includes the induction of cell motility, repression of the cytokeratin expression, and activation of vimentin expression.

Snail and Slug are known to regulate the expression of isoforms of another transcription factor p63 that is required for proper development of epithelial structures. The altered expression of p63 isoforms reduced cell–cell adhesion and increased the migratory properties of cancer cells. The p63 factor is involved in inhibiting EMT and reduction of certain p63 isoforms may be important in the development of epithelial cancers. Some of them are known to regulate the expression of cytokeratins.

Recently, activation of the phosphatidylinositol 3' kinase (PI3K)/AKT axis is emerging as a central feature of EMT.

Twist, another transcription factor, has also been shown to possibly induce EMT, and is also implicated in the regulation of metastasis. Expression of FOXC2, an important player during embryonic development has been shown to induce EMT and regulate metastasis. Moreover, expression of FOXC2 is induced when epithelial cells undergo EMT by Snail, Twist, Goosecoid, and TGF-beta 1.

EMT may be induced by type I collagen, mediated by integrin α1β2. As cells assume a more mesenchymal phenotype, expression of molecules such as osteopontin and type 1 collagen are increased.

Role in metastasis and proliferation
Initiation of metastasis involves invasion, which has many phenotypic similarities to EMT, including a loss of cell-cell adhesion mediated by E-cadherin repression and an increase in cell mobility. Loss of certain genes (e.g. Hedgehog family) has been shown to activate integrin, Wnt, and possibly other signaling pathways, leading to alterations in cell-cell adhesion.

EMT is a characteristic feature of cells undergoing proliferation. Cells expanding in-vitro, like beta cells- and epithelial phenotype, of the pancreas, assume mesenchymal phenotype. Similarly cultured hepatocytes and kidney tubular epithelial cells undergo dedifferentiation in a process similar to an EMT event. In-vivo (via KO or under cancer-inducing scenarios), EMT has been shown to occur in proliferating cells (e.g. stomach epithelium) when pathways known to be involved with EMT are altered.

Nicotine may contribute to EMT. Molecular factors that participate in EMT-related processes include also Hedgehog, nuclear factor-kappaB and Activating Transcription Factor 2.

The concept of epithelial–mesenchymal transition (EMT) was also demonstrated to be useful in generation of endocrine progenitor cells from human pancreatic islets. However, there has been significant debate in understanding the proliferative potential of "terminally" differentiated cells, such as the insulin-producing β-cells. The entire debate started after the initial presentation of EMT in cadaveric human islets. These investigators proposed that human islet-derived progenitor cells (hIPCs) are better precursors since β-cell progeny in these hIPCs inherit epigenetic marks that define an active insulin promoter region. Although similar observations in single cells obtained from human islets were also reported shortly after this initial presentation, the entire concept was strongly opposed by a series of articles. These researchers used genetic lineage tracing system to label β-cells and convincingly demonstrate that labelled (mouse) cells do not exist in the expanded (proliferating) cultures. Two of these articles noted that labelled β-cells de-differentiate to a mesenchymal-like phenotype in vitro, but fail to proliferate. Overall, these articles, suggested that (mouse) β-cells do not proliferate /undergo epithelial-mesenchymal transition (EMT) in vitro. Since previous studies in human islets lacked lineage-tracing analysis, these findings from irreversibly tagged beta cells in mice were extrapolated to human islets. It became a consensus that terminally differentiated islet β-cells do not proliferate in vitro and the mesenchymal population seen in vitro was proposed to arise from rapid proliferation of pre-existing mesenchymal cells. However, the group of Shimon Efrat, used a dual lentiviral system to irreversibly label human β-cells in vitro, demonstrating that adult human islet β-cells undergo EMT and proliferate in vitro. Following this publication, the group of Anandwardhan Hardikar published data confirming these findings in human fetal pancreatic insulin-producing cells. These authors used multiple approaches, including immunostaining and FISH, single cell PCR, clonal expansion analysis, assessment of heritable marks of insulin-promoter region and thymidine-analog based lineage tracing analysis to demonstrate proliferation of human fetal insulin-producing cells. Furthermore, the same group also demonstrated that members of the miR-30 family of microRNAs (a class of non-codingRNAs) are involved in regulation of EMT in human islets, mainly due to the genomic (intronic) location of members of this family. These studies from the group of Efrat and Hardikar now confirm that human pancreatic insulin-producing cells proliferate and undergo EMT in vitro. These groups have also indicated that mesenchymal cells derived from pancreatic islets can undergo reverse EMT or mesenchymal–epithelial transition (MET) to generate islet-like cell aggregates. Although such islet-like aggregates show very low levels of insulin, the concept of generating progenitors from insulin-producing cells by EMT may help in generation of lineage-committed islet progenitor cells. Such cells may have potential for replacement therapy in diabetes.