Epithelial to mesenchymal transition

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The term ‘epithelial to mesenchymal transition' was first coined in 1987 by Krug, Mjaatvedt and Markwald (Krug et al., 1987) in order to describe cellular changes brought about by the extracellular matrix, but it was until 1995 that epithelial to mesenchymal transition was fully elucidated by Hay (Hay 1995). EMT can be defined on the basis of both functional modifications that cells undertake, as well as the associated molecular characteristics.

Functional characteristics of epithelial-mesenchymal transition

Epithelial cells are comprised of sheets of tightly packed immobile cells that are strongly polaraized along the apical-basal axis, reflecting their function as structural junctions between neighbouring tissues of different types, which is a vital requirement for embryogenesis. With increasing complexity of the developing embryo of many Metazoan organisms, polarity changes allow epithelial cells to escape the surrounding fixed tissue and are internalised to form migratory mesenchymal cells of the newly developing mesoderm . Epithelial-mesenchymal transition involves a cellular switch from the powerful cell-cell adhesion of epithelial cells to a more elongated mesenchymal cell with increased capabilities for migration and invasion and is somewhat a reversible process (mesenchymal-epithelial transition) that reflects the ever changing environmental cues that a cell must respond to during embryogenesis (Hugo et al., 2007). EMT is central in development as the motile nature of the resulting mesenchymal cells ensures the correct development of a diverse array of tissue structures such as the muscular system, bone, connective tissue, heart valve and neural crest are also orchestrated by the process of Epithelial-mesenchymal transition (Blick et al, 2008).

Molecular markers of epithelial-mesenchymal transition

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Many molecules appear to participate in achieving epithelial-mesenchymal transition. EMT arises due to the disappearance of cellular junctions brought about by the transcriptional repression of E-cadherin, resulting in the loss of cell-cell adhesion. This process is accompanied by an alteration in cytoskeleton arrangement through the up-regulation of proteases. F-actin fibres of the cytoskelelton are replaced by a network of proteins that contribute to the mesenchymal phenotype such as vimentin and N-cadherin (Thiery, 2000). Initially believed to be a molecular marker of epithelial- mesenchymal transition, vimentin is now considered to have a more functional role in cells demonstrating mesenchymal properties (Nieman, 1999).

A relocation of cell organelles as well as the induction of machinery necessary for cell migration is generated by Rho small GTPases (Guarino, 2007). Other examples of molecular markers of EMT include an increase in expression of transcription factors such as constituents of the ZEB, snail and helix-loop-helix families (Thiery & Sleeman, 2006).

Tyrosine or serine-threonine kinase receptors that operate at tumour-stroma junction, ECM and related molecules such as integrins, matrix-degrading and collagens as well as oncogenic signal tranduction pathways cooperate in the progression of EMT. Under normal conditions, growth factors located in the stroma along with its associated collagens never come into contact with the epithelium but in doing so could induce EMT (Guarino, 1995).

1.2 Epithelial to mesenchymal transition in Development

The original epithelial to mesenchymal transistion that occurs in the developing embryo involves the construction of the primary mesenchyme from the upper epiblast epithelium to form a blastocyst consisting of the three germ layers; ectoderm, mesoderm and endoderm (Duband et al., 1995). The first somite is formed by a process of mesenchymal to epithelial transition and is closely followed by a second EMT event that takes place in the ectoderm and results in the creation of neural crest cells which migrate to make the peripheral nervous system. Further epithelial to mesenchymal transitions shape the secondary mesenchyme from the ventral somite, and muscle and connective tissue of the skin's dermal layer from the dorsal somite. Epithelial to mesenchmal events that occur during development are summarised in figure 1.

However epithelial to mesenchymal transition is not restricted to the events of embryogenesis. Placental development, as well as the healing of a wound are examples of other processes orchestrated by EMT. There is also evidence demonstrating that in adults EMT contributes to tissue fibrosis through the formation of fibroblasts (Guarino, 1995)

These, like many other events regulated by EMT, are under the stringent direction of growth factors as well as many transcription factors acting downstream (Hugo et al, 2007).

Molecular Mechanisms of Epithelial Mesenchymal Transmission

Discovery of the molecular basis of EMT

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The molecular mechanisms of the loss of cell polarity and gain of mesenchymal cell properties associated with epithelial to mesenchymal transition were first studied in epithelial cells, and gave rise to the elucidation of numerous transduction pathways involved. (Thiery, 2003).

In 1985, Michael Stoker and Michael Perryman first uncovered evidence for the molecular basis of epithelial to mesenchymal transition when they observed that Madin-Darby canine kidney (MDCK) cells in the supernatant of a fibroblast culture were induced by a factor to undergo separation at cellular junctions, resulting in a scattering of cells. Stoker and Perryman found that following the addition of the factor, separation was induced after only 15 minutes and triggered the migration of MDCK cells into wounds. Although a physiological role of this factor had not yet been recognised, Stoker and Perryman had correctly proposed a role of this scatter factor in cell migration (Stoker and Perryman, 1985). This work stimulated others to enter the field of study of EMT, and in the 1990s the scatter factor described by Stoker and Perryman in the 1980s was finally ascertained to be the hepatocyte growth factor, a ligand of the c-met receptor. Since then many other growth factors recognising and binding to tyrosine kinase receptor have been discovered that are now known to induce epithelial to mesenchymal transition.

Discovery of signal transduction pathways

Many complex and overlapping signal transduction pathways form the basis of epithelial-mesenchymal transition both developmentally and during carcinoma invasion. Growth factors such as hepatocyte growth factor, epithelial growth factor (EGF) fibroblast growth factor, and insulin-like growth factors 1 and 2 have been shown to induce epithelial to mesenchymal transition in vitroin numerous epithelial cell lines by means of activation on binding to tyrosine kinase receptors. (Thiery, 2002).

Tyrosine kinase receptors

Tumour invasion

Epithelial-Mesenchymal transition has long been coupled with tumour invasion and the advancement of breast cancer into eventual metastasis but is at present still not fully understood. Tumours consist of fixed, highly organised and tightly linked epithelial cells. E-cadherin establishes these connections between cells that initially stay bound to the basement membrane in a fixed array. In order for these cancerous cells to invade other tissues, they must conquer the strict cell-cell interactions of the basement membrane to gain entry into the stroma via the process of epithelial to mesenchymal transition (Thiery and Sleeman, 2006).

Epithelial to mesenchymal transition enables the translocation of these tumour cells and infection of adjacent tissues through the deterioration of intercellular adhesion proteins that are dependent on E-cadherin. The elimination of E-cadherin is brought about through signal transduction cascades, beginning with the binding of growth factors to receptors on the cell membrane. The loss of cellular junctions allows the cytoplasm to extend out of the cell through fractures in the membrane as projections, permitting the formation of new connections as the contracting cytoplasm forces the cell into the stroma. This simple succession of projection and contraction of tumour cells provides the necessary motile action in which to carry out a successful invasion into neighbouring tissue, and potentially metastasize to outlying organs (Guarino, 2007).

Comparing EMT and tumour migration

Comparisons have been drawn between metastasis and epithelial transmission in development for many years now purely on the basis of migration of mesenchymal cells (Thiery, 2003). In the past, metastasis of cancerous cells was purely evaluated on the basis of migration of cells but other features of disease have recently come to light, greatly increasing the spread of disease to distant organs such as improved cell survival and defiance of apoptosis (programmed cell death) in the face of chemotherapies (Thomson et al., 2005). Tumour invasion begins with the acquisition of cell motility of a solitary cancerous cell, involving disconnection of the single cell from the support system of surrounding epithelia, followed by its translocation into the stromal compartment. This event bears a significant resemblance to the course of action employed during epithelial-mesenchymal transition of the developing embryo (Huber et al., 2006). Down-regulation of E-cadherin directly dictates epithelial-mesenchymal transition in both cancer and development (Guarino, 2007). Molecular techniques may be employed in order to compare EMT with invasion through the targeting of members of the Snail family such as Snail1, Slug, Snail3 as well numerous helix-loop-helix factors like ZEB1, Twist and SIP1 (Peinado et al., 2007).

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Difficulties in proving that epithelium-mesenchymal transition plays a part in the metastasis of breast cancer in a clinical setting has made the characterisation of EMT rather complex and has sparked many debates on the subject. This has led to a new concept of a metasable phenotype, a hybrid condition of incomplete mesenchymal transition seen in cancerous cells (Lee, 2006). Newly transforming mesenchymal cells situated at the invasive front of the tumour must be equipped with the ability to dissociate entirely from the tumour mass, as well as the ability to adapt on reaching an unfamiliar destination in order for complete metastasis to occur (Lee, 2006).

The study of invasive breast cancer cell lines

The examination of invasive breast cancer cell lines can shed more light on the events of epithelial to mesenchymal transition. In the past, metastatic breast cancer was described in terms of an absent oestrogen receptor, but mounting evidence is emerging over the last decade or so through the study of breast cancer cell lines that demonstrate the formation of an intermediate filament protein by way of epithelial-mesenchymal transition cold better explain this phenomenon. Upon further examination it was realised that these invasive breast cancer cell lines had reduced levels, or were completely lacking in cell-cell adhesion molecules such as E-cadherin (Thompson et al., 1992). It was also demonstrated that in cell lines deficient in E-cadherin showed increased levels of N-cadherin, and that breast cancer metastasis was propagated by transfection of N-cadherin regardless of E-cadherin levels (Nieman, 1999).

Markers of EMT in breast cancer cell lines

An interesting study was published last year on the levels of gene expression of familiar EMT markers in basal A, basal B and luminal classes of breast cancer cell lines, encompassing 51 cell lines in total (Blick et al, 2008). Basal breast cancer cell lines are capable of undergoing epithelial-mesenchymal transition and originate from normal human breast cell lines (Peinado et al, 2007). Cell lines of luminal cancer are also capable of EMT in response to oestrogen levels for example (Planas -Silva and Waltz, 2007). The basal A subgroup consist of breast cancer cell lines with properties similar to those of both basal B and luminal breast cancers (Blick et al, 2008).

Common markers of epithelium-mesenchymal transmission utilised in the study by Blick et al included E- and N-cadherin, vimentin and fibronectin. From the data in the figure below obtained from Blick's paper it is evident that E-cadherin expression levels are highly reduced while N-cadherin levels are greatly increased in Basal B cell lines in comparison to both Basal A and luminal cell lines. Recall that in cells, tumour invasion is induced by the up-modulation of N-cadherin in the absence of cell-cell adhesion normally provided by E-cadherin. Fibronectin and Vimentin are highly expressed in both Basal A and Basal B cell lines, but mRNA levels are undoubtedly elevated to a higher degree in Basal B cell lines. It is clearly obvious from the results of this investigation that in the cell lines of the Basal subgroup are indicitve of an EMT phenotype and in order to fully characterise and discover new inducers of epithelial-mesenchymal transition in the future, we must turn our attention to uncovering new EMT markers of the Basal B class of breast cancer cell lines.

MCF10A cell lines

MCF10A cell lines result from spontaneous epithelial-mesenchymal transition of cells in response to the up-regulation of epidermal growth factor (EGF) following mastectomy or breast reconstruction surgery (Gilles, 1999). Elevated levels of NF-kB as well as snail-mediated repression of E-cadherin in MCF10A cell lines serves as a marker for EMT induction via the up-regulation of type one insulin-like growth factor (IGF-IR) (Kim et al., 2007). The subunit p65 is constitutively functional in IGF-IR, and its addition to MCF10A cell lines results in an EMT phenotype related to expression of the oncogene ZEB-1. Tumour necrosis factor alpha (TNFα) can stimulate the production of NF-kB which also initiates EMT through the activation of ZEB-1 or ZEB-2 (Chua et al., 2007).

Development of innovative anti-cancer therapies

There is a tremendous demand for the development of novel therapeutic strategies in the treatment of breast cancer. A major incentive for this is the inefficiencies of modern chemotherapies which are not only poisonous to the body, but are often non specific and can acquire resistance over time (Shah and Gallick, 2007). Hope for potential anti-cancer therapies rests in the analysis and targeting of precise molecules in signal transduction pathways. Since epithelial-mesenchymal transition begins with the migration of a solitary cell, knowledge of the molecular basis of EMT could provide improved therapies with increased specificity with the ability of targeting the individual cells before getting the opportunity to invade surrounding cells and therefore blocking possible future metastasis.

As snail plays such a pivotal role in the control of epithelial-mesenchymal transition, targeting upstream molecules that modulate snail's stability and localization within the cell such as PAK (a positive regulator of snail), GSK-3B (a negative regularor of snail) and TGFB would prove to be a useful strategy to impede EMT (Peinado & Cano, 2006). Methods which could be employed in order to direct the molecular targeting of snail include the silencing of expression of the snail gene through the application of small interfering RNA (siRNA) or by the obstruction of formation of snail's protein product by chemical means (Guarino, 2007). The discovery of novel pathway-specific treatments may prove arduous as epithelial-mesenchymal progression most likely involves a combination of many overlapping pathways.

Discussion

The precise events of epithelial to mesenchymal transition ultimately leading to metastasis of distant organs is still ambiguous despite such a significant clinical importance but the advancement of profiling has led to the detection of a growing number of molecular markers of EMT (Blick, 2008). Enhanced analytic techniques of an ever increasing number of cell lines have greatly benefited investigations into breast cancer metastasis (Blick, 2008). Proteins down regulated during epithelial to mesenchymal transition that are linked to tumour invasion could be employed as markers for detection of cancer or in therapeutics.