Epithelialmesenchymal transition

Epithelial cell structure is maintained by cell-cell interactions involving tight junctions and desmosomes and these cells are non-motile. In contrast, mesenchymal cells do not have cell-cell contacts but have distinct cell-ECM interactions and cytoskeletal structures and are motile. Epithelial-mesenchymal transition (EMT) is a series of events where the cell-cell and cell-ECM interactions are altered resulting in detachment of epithelial cells from the sur rounding tissue followed by rearrangement of the cytoskeleton to confer the ability to move through a three-dimensional ECM and the induction of a series of new transcriptional signaling pathways to maintain the mesenchymal phenotype [42]. This process is important in embryonic development, particularly in gastrulation and segment formation. However, more recently, EMT has been implicated in carcinogenesis. EMT involves a multistep process in which the non-motile epithelial cells are tranformed into motile invasive cells [43]. This process is quite similar to the onset of the invasive metastasis process where there is a transition from a benign to aggressive tumour phenotype, involving the detachment of tumour cells from the primary site followed by invasion through the ECM (Figure 1). The reverse of the EMT process is known as mesenchymal-epithelial transition (MET), which facilitates tumour cell attachment at secondary sites.

Integrin

Integrin expression

Reference

a2ß1

4 Prostate hyperplastic tissue t Metastatic prostate cancer tissue t Metastatic prostate cancer cell lines

Bonkhoff et al. (1993) Hall et al. (2006), Bostwick DG et al. (2006)

a3ß1

t Associated with higher recurrence

Pontes-Junior et al. (2010),

aiibß3

t Metastatic prostate cell lines

Trikha et al. (1998)

a4

4 Metastatic prostate cencer cell lines and xenograft samples

Saramaki et al. (2006)

a7

4 Metastatic xenograft samples

Ren et al. (2007)

avß3

t Prostate cancer tissue samples, metastatic prostate cancer cell lines

Zheng et al. (1999), Nameth et al. (2003)

a5ß1

t Metastatic prostate cancer cell line

Stachrurska et al. (2012)

a6ß1

t Metastatic prostate cancer tissue and metastatic prostate cancer cell lines

Davis et al. (2001), Bonkhoff et al. (1993), Trikha et al. (1998), King et al. (2008)

a6ß4

4 Metastatic prostate cancer tissue

Davis et al. (2001)

Table 2. Integrin expression in prostate cancer progression

Table 2. Integrin expression in prostate cancer progression

EMT involves a series of signalling processes. Firstly, it involves the break-down of cell-cell interactions leading to loss of E-cadherin expression and the upregulation of mesenchymal markers such as N-cadherin, vimentin and the transcription factors Snail, TWIST and ZEB family members. Then, it is followed by a loss of cell polarisation and cytoskeleton remodelling. Finally, changes in cell adhesion occur leading to cell detachment and the activation of proteolytic enzymes; matrix metalloproteinases (MMPs) [44]. The initiation of EMT is tissue and context dependent and may not involve all EMT markers [45]. There are various stimuli from outside the cell which regulate EMT within the tumour microenvironment. These include the binding of transforming growth factor-p (TGFp) to the TGFp receptor (TGFpr), growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) which bind to the tyrosine-kinase receptor (TKR), the highly conserved Wnt/ß-catenin pathway and also integrin signalling which activates the FAK signalling pathway [46, 47]. Since integrins are involved in cell adhesion and signalling, it is possible that integrins can initiate and mediate EMT and invasion in tumour progression (Figure 1).

E-cadherin is a type-I cell-cell adhesion glycoprotein and is a major inducer of EMT as loss of E-cadherin results in decreased cell adhesion and thus, increased cell motility. It is expressed by most epithelial tissues and it forms the tight junction connecting adjacent cells and thus, the formation of stable cell-cell contact. Loss of E-cadherin has been associated with tumour progression and metastasis in breast cancer, prostate cancer, colorectal cancer and gastric cancer [44-48]. Besides genetic and epigenetic factors, transcription factors such as the zinc finger proteins, Snail, Slug, ZEB1, ZEB2 and the basic helix-loop-helix protein, TWIST are involved in the repression of E-cadherin. The zinc finger proteins repress E-cad-herin by binding to the E-box motif in the E-cadherin promoter. The role of the Snail transcription factor on E-cadherin has been studied in epithelial tumour cell lines of different origins including bladder cancer, pancreatic cancer and colon cancer. Most of the cell lines showed an inverse correlation between E-cadherin and Snail expression levels and when Snail was transfected into the cell lines that express high E-cadherin levels, it resulted in down-regulation of E-cadherin [49]. It has been proposed that Snail and ZEB2 initiate the silencing of E-cadherin by modifying chromatin organisation of the gene [50]. Subsequently, Slug and ZEB1 have been proposed to be responsible for maintaining the repression of E-cadherin and thus, maintenance of the mesenchymal phenotype [51].

TGFß signaling is the main inducer of EMT in the development of cancer. Interestingly however, the TGFß response is context-dependent where it can either act as a growth inhibitor or it can induce tumour progression by promoting angiogenesis, immune suppression and preventing apoptosis [52, 53]. The main role of TGFß is to induce apoptosis and thus, it generally acts as a tumour suppressor during the early stages of cancer progression. However, frequent loss-of-function mutations in TGFß have been observed in cancer, which is associated with the progression of cancer by inducing cell metastasis. Multiple signalling pathways are involved in the induction of EMT by TGFß including the Wnt/ß-catenin pathway and integrin signalling pathways. Ligation of the TGFßr results in the activation of Smad2 and Smad3 and constitutive phosphorylation of Smad4 [54]. These Smads then bind to ZEB1 and ZEB2 to repress E-cadherin expression [55-57]. Miyaki et al. (1999) found increased mutations in Smad 4 as the stage of colorectal tumours advanced, suggesting that inactivation of Smad 4 in the TGFß signalling pathway induces tumour metastasis [58].

Activation of the TKR by growth factors has also been found to induce EMT. Stimulation of the breast cancer cell line, PMC42-LA with EGF resulted in E-cadherin downregulation and upregulation of vimentin expression [59]. This is followed by increased cell adhesion and migratory capacity suggesting the upregulation of integrins upon EGF treatment. Integrins have also been linked to EMT as discussed below.

Collagen

Fibronectin

Collagen

Fibronectin

:

Epithelial cell

M

Mesenchymal cell

E-cadherin

ff

Integrin

II

TGFßr

II

TKR

II

Wnt

Figure 1. Schematic representation of the EMT process and the roles of integrins in cell adhesion and migration

6. Roles of integrins and EMT in cancer

To date, studies on the involvement of integrins in EMT during cancer progression have been limited, particularly in prostate cancer. Here we highlight the recent studies which correlate integrins (implicated in prostate cancer) and EMT. EMT involving changes in the expression of cadherins has been observed in prostate cancer progression [44]. Loss of E-cadherin (epithelial marker) expression has been correlated with increased tumour grade, with 46 out of 92 prostate tumour samples showing reduced or absence of E-cadherin staining when compared to non-malignant prostate samples [45]. In contrast N-cadherin (mesen-chymal marker), was not expressed in normal prostate tissue but expressed in the poorly differentiated areas of prostate cancer specimens, where E-cadherin was absent [44]. These studies suggest that switching of cadherin expression correlates with prostate cancer metastasis.

Collagen type I which is the ligand of integrin a2p1 was found to induce the disruption of E-cadherin adhesion complexes in pancreatic cancer [60]. The study suggested that binding of collagen type I to a2p1 activates FAK phosphorylation which enhances tyrosine phosphorylation of p-catenin and causes the disassembly of the E-cadherin complex. In addition, Shintani et al. (2008) showed that activation of integrin a2p1 by collagen type I together with activation of the discoidin domain receptor 1 (DDR1) induces N-cadherin expression [61]. Furthermore, high E-cadherin was observed in suspended PC3 cells and the expression decreased as cells attached to a fibronectin substrate, whereas N-cadherin expression was 4fold lower in suspension cells compared with attached cells [62]. Blocking of the integrin p1 by the AIIB2 antibody resulted in no increase of N-cadherin expression in PC3 cells, suggesting that integrin p1-mediated cell adhesion to fibronectin is involved in regulating N-cadherin expression in prostate cancer. The study also investigated the regulation of N-cadherin by Twist1 (a transcription factor that regulates mesenchymal gene expression). Knockdown of Twist1 expression in PC3 cells resulted in decreased N-cadherin expression and inhibition of cell migration. Interestingly, blocking of integrin p1 correlated with inhibition of nuclear accumulation of Twist1 following cell attachment. Therefore, these data suggest that the integrin p1-mediated adhesion is regulated through Twist1 accumulation and activation of N-cadherin.

Integrins have also been shown to activate latent TGFp. TGFp is involved in tissue homeo-stasis and is both a tumour suppressor and tumour inducer, as outlined above. Tumour cells have increased secretion of TGFp which induces EMT [63]. Studies have found that TGFp can be activated by integrins. Bates et al. (2005), developed a colon cancer model of EMT, where EMT can be induced in the LIM 1863 colon cancer cell line by exposure to TGFp. This model showed that EMT resulted in upregulation of integrin avp6. This occurs through the Ets-1 transcription factor and integrin avp6 was found to promote the activation of autocrine TGFp in post-EMT to stabilize and sustain EMT and also promote cell migration on fibronectin [64]. In another study, in order to study the role of TGFp, stable clones of truncated TGFp were generated in non-transformed mouse mammary ductal epithelial cells (NmuMG) [65]. The truncated TGFp resulted in blocking of TGFp-mediated growth inhibition, Smad-mediated transcriptional activation, AKT signaling pathways and EMT. However, this did not block the TGFp-mediated p38MAPK activation. Further, blocking of integrin p1 with antibody resulted in inhibition of p38MAPK and EMT progression. Therefore, these results suggest that TGFp-induced EMT is dependent on both p38MAPK activation and integrin p1 which thus suggests the cooperation of TGFp and integrins in the modulation of EMT progression.

The mesenchymal transcription factor Snail plays a role in EMT by repressing E-cadherin. A study investigated the regulation of integrin av expression by Snail in epithelial Madin-Dar-by canine kidney (MDCK) and A431 cells [66]. Upregulation of integrin av was observed in MDCK Snail transfected cells and A431 Snail transfected cells. Further investigation showed expression of integrin av was mediated directly through its promoter by the Snail transcription factor. In addition, MDCK Snail transfected cells showed increased cell migration towards osteopontin, the ligand for integrin avß3 in bone. Therefore, these data suggest that Snail enhances cell migration, at least in part, by regulating integrin expression in cells. A more recent study which involved stable transfection of Snail into ARCaP and LNCaP prostate cancer cell lines, found a decrease in cell adhesion and increase in cell migration on collagen I and fibronectin [67]. The Snail transfected ARCaP cells were then subjected to flow cytometry and results showed downregulation of integrin a5, a2 and ß1, which was reversed by Snail knockdown.

A microarray study undertaken to examine 19 primary prostate tumours showed 65% loss of E-cadherin in metastatic tumour samples compared to primary tumours [18]. The expression levels also correlated with a 71% loss of integrin ß4 when comparing metastatic to primary tumours. These results suggest that progression of prostate cancer involves the loss of E-cadherin and a possible involvement of E-cadherin in regulating integrin ß4 expression. More recently, a study has found expression of ZEB1 which is a dual zinc finger transcription factor and a known regulator of EMT to repress integrin ß4 expression in PC3 cells [68]. Further, transient transfection of ZEB2 in the colon cancer cell line, SW480 was found to up-regulate the expression of integrin a5 [69]. Knockdown of ZEB2 resulted in suppression of integrin a5 and the cells displayed reduced cell invasion. In addition, ZEB2 was found to cooperate with the SP1 transcription factor to activate the integrin a5 and vimentin promoters and thus, induction of the mesenchymal gene during EMT in cancer progression.

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