Epithelial to mesenchymal transition in the cancer microenvironment

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The integrity of the tissue and its functions are enabled by proper cell-to-cell contact and communication. The importance of cell-to-cell adhesion is illustrated by the phenomenon of anoikis - that is, cell death caused (97) by the loss of interaction with other cells or between the cell and its microenvironment. This phenomenon therefore indicates that cell adhesion impacts cell survival  (98-99). In vivo normal cells detached from the microenvironment are not able to home other tissues or to develop into an ectopic growth. Unlike normal cells, however, cancer cells become resistant to anoikis because of the various alterations both in adhesion protein expression and in the interactions between cells. The expression of proteins regulating apoptosis facilitates the survival of detached single cells and so their survival in ectopic localizations (100). The creation of distant metastases is preceded by a disturbance in cell-to-cell adhesion as well as the loss of the integrity of the primary tumor (101-102). The proteins responsible for the intracellular communication are cell adhesion molecules (CAMs). These are glycoprotein receptors that constitute the integral part of the cell membrane  (103) . CAMs condition the interactions between cells, their exchange of information, their control, and their localization in the proper tissue. Among the members of the family of CAMs, the most important are immunoglobulin superfamily proteins, selectins, integrins, adresins (CD34, Gly-CAM-1, MAdCAM-1), cadherins, and CD44 molecules (103). Cadherins are glycoproteins with two domains, transmembrane and extracellular. The extracellular domain has a calcium-binding locus, which is a site for binding with another molecule from the same class (104) to form a mechanical junction of adherence. Cadherins bind to the actine cytoskeleton of another cell through the adhesion complex including catenins (-, β-, - and p120-catenin). They also participate in signal transduction (105) and their main role is to create intracellular junctions. E-cadherin is the most important protein for cell polarization and the organization of the epithelium. In various types of malignant epithelial neoplasms, the intracellular adhesion carried by E-cadherin disintegrates as the tumor progresses and correlates with tumor grade and a poor prognosis (106) . Intermediate filaments are the chief components of the cytoskeleton; they seem to play an important biological role given their abundance and because expression changes correlate with alterations in cell behavior. Among the intermediate filaments, four groups of proteins are selected with respect to their structure, including: type I-keratins, type II-cytokreatins, type III-vimentin, and type IV-neurofilaments. Vimentin is a 54 kDa protein connected with cell organelles, elements of cytoskeleton, and membrane adhesion factors which reflect the integrity of vimentin in their cell structure and function. Vimentin is present in various types of cells, including fibroblasts, endothelial cells, macrophages, neutrophils, and lymphocytes. It plays many important biological roles in physiological development and is expressed in adult mesenchymal cells of the central nervous system and muscles (107). Vimentin expression was observed in rat’s and monkey’s testes in Sertoli’s cells where the level decreased with age, leading to weakened vimentin filaments. This in turn resulted in the impairment of the function of these cells and a disturbance in Sertoli cell development (108). Vimentin overexpression accompanies immune-mediated diseases and graft rejection. Vimentin expression levels have also been observed to be significantly increased in the endothelium of patients following the renal graft rejection in comparison to those patients in whom the graft was not performed. Vimentin is constitutively expressed in endothelial cells; it might be overexpressed in response to stress during the procedure of renal graft or following the graft rejection (109). As has been shown, vimentin is related to cholesterol transport during the steroidogenesis (107). It facilitates the development of the placenta and trophoblast invasion and its overexpression has been observed in invasive cells during placental development (107). The accumulation of vimentin in cells is a highly organized and dynamic process. It has been shown that vimentin may not only be apparent in the form of filaments, but also in non-filamental forms, unconnected with the membrane form of aggregates in cell cytoplasm, changing their shapes constantly, connecting and detaching, shortening and becoming longer (107, 110-112). In sum, it must be stressed that an increase in the level of vimentin expression correlates in many studies with an increase in cell migration and invasive properties both in physiological and pathological situations, such as in malignant neoplasms. Additionally, vimentin is a mesenchymal marker of the EMT process (107).

The phenomenon of epithelial to mesenchymal transition is characterized on the molecular level by changes in the expression of epithelial markers and by an increase in proteins related to migration and invasion. In this way, the metastasizing cells share many similarities with the cells undergoing EMT, and it is thought that EMT participates in the progression of cancer. EMT is defined as a situation where a cell loses its stable, polarized, non-migrative properties and takes on fibroblastic, migrational abilities with typical mesenchymal features. Such cells lose their polarization, and alterations in the cell-to-cell and cell to the extracellular matrix adhesion lead to their increased mobility (107).

EMT occurs physiologically in such tissue processes as embryonic development, tissue remodeling, wound healing, and inflammation (113). Various in vitro studies have indicated that both morphologically and on the molecular level, the changes in cancer cells necessary at the early stages to create metastases mimic the physiological EMT process  (113). The factors inducing the EMT process are the growth factors secreted by cancer cells and their microenvironment: fibroblast growth factor-2 (FGF-2) and transforming growth factor-β (TGF-β). During EMT, epithelial cells progressively redistribute and decrease the expression of proteins specific for the apex and basis of the cell, such as adhesion molecules, including E-cadherins. The re-expression of mesenchymal proteins, such as vimentin and N-cadherin, has also been observed. These changes lead to the disintegration of intracellular junctions and the acquisition of the cell mobility needed for invasion (114).

The gene for E-cadherin is a tumor-suppressor gene inhibiting the creation of metastases. The inhibition of E-cadherin expression is observed during transcription through transcription-inhibiting factors (Snail1, Snail2 and others) (1115-116). The expression of inhibiting factors is regulated by TGF-β, FGF, EGF, Stat3, and NFkB (117-118). As a result of transcription suppression, factors inducing E-cadherin expression are recognized in various cancer cells under hypermethylation status (119). E-cadherin can also be inhibited at a protein level. The tyrosine kinase receptors, such as epithelial growth factor receptor (EGFR), c-Met, insulin-like growth factor receptor 1 (IGFR-1), and fibroblast growth factor receptor (FGFR) induce the phosphorylation of E-cadherin and catenins, leading to their degradation(120-121). Other factors, such as proteases, MMP-9, TGF-β, and HGF/SF are able to cut E-cadherin and destroy the intracellular contacts that it mediates (122). The most recent studies show that mesenchymal cadherins, especially N-cadherin, increase cancer cell motility and their migration counters E-cadherin action (123). In fact, cancer cell invasion induced by N-cadherin may exceed the pro-adhesive E-cadherin activity. These observations would seem to confirm the existence of a cadherin switch stimulating the change from epithelial into mesenchymal cadherins and encouraging the transformation from non-invasive to invasive tumor phenotype. Moreover, it has been shown that N-cadherin increases the ability of tumor cells to migrate by inducing the transduction of signals through the stimulation of FGF receptor (121-122).

It has been established that EMT may be induced in cancer cell lines in response to such growth factors as HGF, TGF-, and EGF (123). The transition to the mesenchymal phenotype is related to more aggressive tumor behavior and a higher degree of invasiveness. Squamous-cell carcinoma cells obtained from metastatic cells in lymph nodes in vitro exhibited greater proliferative ability and motility than the primary tongue cancer cells in vivo. It was shown that cancer cells obtained from the metastatic lymph nodes had a higher level of vimentin expression than that observed in the primary cancer cells. Moreover, the vimentin expression level increased under EGF and TGF- factors when applied either together or independently. Cancer cells with lower levels of vimentin expression had decreased proliferative potential and were about 70% smaller than the control cells. These findings indicate that it might be possible to reverse the mesenchymal phenotype of cells by blocking vimentin expression, which causes the re-expression of epithelial markers and results in lower tumor aggressiveness (124). Moreover, it was confirmed that active Src (a potential factor inducing the EMT process; while tyrosine kinase is the aim for growth factors), together with decreased E-cadherin and increased vimentin expression levels, participated in inducing EMT transformation (125). In head and neck cancer, a correlation between the invasive type of growth and poorly differentiated cancers was noted (125-126). The EMT phenomenon in this study was determined by the high level of vimentin expression, low level of E-cadherin expression, and high degree of Src tyrosine kinase activity. In our studies, vimentin expression was observed in both head and neck squamous-cell carcinoma and its microenvironment and it characterized the invasive, mesenchymal phenotype. It was furthermore observed that the vimentin expression level increased with tumor size; no correlations, however, were observed between the vimentin expression level and the presence of lymph node metastases or the tumor grade (69-70).

In sum, tumor-associated macrophages and cancer-associated fibroblasts, expressing RCAS1, B7-H4 molecules, and MT participate in creating the suppressive profile of the cancer microenvironment and in the cancer microenvironment remodeling that enables both local tumor spread and the creation of metastases.