1.3 EMT IS ESSENTIAL FOR MAMMARY GLAND DEVELOPMENT, AND DISTURBANCE OF
1.3.1 The EMT process is regulated by several signalling pathways that implies the
stimulation from the microenvironment
Several pathways are involved in the regulation of EMT in human tissue including Hedgehog, TGF-b, Notch, Wnt/b-catenin, TNF-a, integrins and receptor tyrosine kinase pathways (Garg, 2013) (Figure 0-2). Upon activation of the receptors with ligands, a signalling cascade is triggered inside the cells and initiates the process of EMT.
Figure 0-2, Pathways involved in the regulation of the process of EMT.
The signalling pathways involved in regulating EMT. (A) Transforming growth factor b (TGFb) signalling is induced by binding of ligands to receptor. TGFb receptor activation by phosphorylation results in recruitment of SMAD transcription factors that translocate to nucleus and binds to DNA (Xu et
al., 2000; Gonzalez and Medici, 2014). (B) Integrins are heterodimeric transmembrane proteins. Integrin
interact with TGFb and activate the pathway (Mamuya and Duncan, 2012) also, integrins interacts with components of extracellular matrix (Yen et al., 2014). (C) Receptor tyrosine kinases (RTKs) are activated by binding to growth factors including epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF). The binding results in dimerisation of receptors and eventually activation of signalling cascade (Lemmon and Schlessinger, 2010; Gonzalez and Medici, 2014). (D) Wnt signalling is transduced by Wnt binding to Frizzled receptor or low-density lipoprotein receptor-related protein (LRP) receptors. Wnt activation releases b-catenin which translocates to nucleus and binds to T-cell factor/lymphoid enhancer binding factor-1 (TCF/LEF- 1) (Gonzalez and Medici, 2014; Nusse and Clevers, 2017). (E) Notch receptor is composed of extracellular and intracellular domain. Upon binding g-secretase cleaves intracellular domain of Notch (NICD) that translocates to nucleus (Kopan, 2002; Miele et al., 2006; Gonzalez and Medici, 2014). (F) Ligands of Hedgehog signalling pathway include Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh) that bind to patched homolog 1 (Ptch1) and Ptch2, which release inhibition on Smoothened (Smo) (Gallet and Therond, 2005; Gonzalez and Medici, 2014). (G) The inflammatory stimuli activate TNFa and IL-6 which activate transcription factor signal transducer and activator of transcription 3 (STAT3), that activates Snail expression. (H) Hypoxia influence EMT by activation of Twist expression after activation of hypoxia-inducible factor 1a (HIF1a) (Yang et al., 2008a). The figure was drawn by the author from the mentioned references.
Several layers of crosstalk between the EMT pathways have been identified. This crosstalk is due to the diverse range of processes involving EMT (Gonzalez and Medici, 2014). The context of cross-interaction between these signalling cascades also depends on interactions with the surrounding microenvironment and the cell state (Gonzalez and Medici, 2014).
The cellular statuses that result in triggering the EMT process include hypoxia (Lundgren et al., 2009; Daly et al., 2017); activation TGFb and other growth factors (Daniel et al., 1996; Cheng et al., 2012); autocrine feedback loops (Schlange et al., 2007); and extracellular matrix remodelling (Silberstein et al., 1992; Gonzalez and Medici, 2014).
Zeisberg and Neilson (2009) categorised the biomarkers that identify the various types of EMT into five groups; cell surface markers (E-cadherin), cytoskeletal markers (vimentin), extracellular matrix proteins (collagen and fibronectin), transcription factors (Snail, Slug, and Twist), and micro-RNAs (Figure 0-3).
Figure 0-3, Markers of EMT.
The types of markers that are affected by EMT with few examples. (A) Changes in cell surface markers including attenuated expression of E-cadherin and change of N-cadherin level. (B) Changes in cytoskeletal markers such as attenuated expression of cytokeratin; changes in localisation of b-catenin; changes of mesenchymal markers level such as vimentin and a-smooth muscle actin. (C) Changes of extracellular matrix proteins such as collagen and fibronectin. (D) Changes of EMT transcription factors expression such as; Snail 1 (Snail), Snail 2 (Slug), ZEB 1, Twist and LEF-1. (E) Changes of microRNAs expression. The figure was drawn by the author and details adapted from (Zeisberg and Neilson, 2009; Wang and Zhou, 2013).
The most important hallmark of EMT commences with the loss of E-cadherin which directly affects the Wnt/b-catenin pathway. Also, increased expression of the mesenchymal markers (such as vimentin), with changes in the level and localisation of E-cadherin, status of Wnt/b-catenin signalling provides further confirmation of the mesenchymal change.
1.3.1.1 Constant E-cadherin expression and localisation to the cell membrane are the principal determinant of the epithelial status of cells
Cell-cell contact organises the structure of mammary epithelial cells which is mediated by E-cadherin and other membrane proteins (Claudin and Occludin) (Morrow
et al., 2010; Chen et al., 2014). E-cadherin facilitates several cellular functionalities
including cell-cell contact, cellular polarity, differentiation, stem cell properties, cell motility, migration, proliferation and survival. These functions are mediated through interaction with b-catenin and cytoskeletal protein (Figure 0-4-B) (Nagafuchi and Takeichi, 1989; Nieman et al., 1999; Junxia et al., 2010; Chen et al., 2014).
Figure 0-4, E-cadherin structure and the cell-cell adhesion structure formed by E- cadherin.
The figure shows the structure of E-cadherin protein and the cell-cell adhesion that is formed by E- cadherin protein. A- E-cadherin multidomain glycoprotein structure (van Roy and Berx, 2008). B- The cell-cell calcium-dependent adhesions formed by E-cadherin assembly to the actin cytoskeleton through catenin proteins. It is linked to the cytoskeletal filaments via interaction with cytoplasmic proteins including (a-catenin and b-catenin) (Yoshida and Takeichi, 1982; Peyrieras et al., 1983). The key below explains the different components of the cell-cell adhesion. The figure was drawn by the author from the mentioned references.
E-cadherin is known as the caretaker of the epithelial phenotype (Thiery, 2002; Oloumi et al., 2004). E-cadherin functions to maintain the epithelial morphology via interaction of extracellular domains, and by intracellular domain binding to catenins (Takeichi, 1991; Tian et al., 2011). Of the components that interact with the intracellular domain of the of E-cadherin, b-catenin is also a crucial regulator of the Wnt signalling pathway (Clevers and Nusse, 2012). Cell contact is a critical determinant of the EMT status of cells, as loss of E-cadherin eventually leads to increasing the free b-catenin pool and, therefore, activation of the EMT process (Hay and Zuk, 1995). Therefore, the loss of cell-cell contact that is mediated by E- cadherin/b-catenin triggers EMT in normal physiological processes as well as in diseases such as cancer (Tian et al., 2011).
In epithelial cells, E-cadherin clusters to the cell membrane and interacts with the E-cadherin of neighbouring cells (Adams and Nelson, 1998; Thiery, 2002). This localisation of the protein to the cell membrane is critical for the maintenance of stable adherens junctions, and disturbance of E-cadherin is known to induce a mesenchymal change (Imhof et al., 1983; Thiery, 2002). In addition, there is a direct correlation between the increase in mesenchymal phenotype and reduction in E-cadherin production (Behrens et al., 1989; Thiery, 2002).
E-cadherin expression in normal mammary gland tissue is strong and localised at the cell boundaries of epithelial cells, forming a cobblestone appearance of epithelial tissue (Wheelock et al., 2001; Kowalski et al., 2003). All non-epithelial tissue in the mammary gland does not express E-cadherin (Kowalski et al., 2003).
Loss of E-cadherin protein or its function as result of mutation is a very rare event in breast cancer (Graff et al., 2000). It was reported previously that the CDH1 (E- cadherin gene) tumour suppressor was lost in ductal carcinoma (Li and Mattingly, 2008; Chen et al., 2014). Graff et al. (2000) concluded that E-cadherin epigenetically silenced was transient and heterogeneous depending on cancer cells microenvironment. They also concluded that loss of E-cadherin expression by methylation in primary breast tumours is an early event (Graff et al., 2000).
E-cadherin is involved in several essential pathways necessary for breast cancer progression. Vutskits et al. (2006) found that E-cadherin has a role in controlling the molecular pathway that regulates the cell cycle. Moreover, disturbance of cell-cell contacts and the cytoplasmic localisation of cadherins stimulate anoikis resistance in
breast cancer cells (Ribeiro et al., 2013). Therefore, loss of E-cadherin is considered as one of the most critical hallmarks of cancer metastasis.
The loss of E-cadherin protein is a hallmark for EMT activation in embryogenesis and cancer progression (Hazan et al., 2000; Huber et al., 2005; Yang and Weinberg, 2008; Polyak and Weinberg, 2009; Thiery et al., 2009; Yuki et al., 2014). Onder et al. (2008) found that complete loss of E-cadherin was essential for cancer progression and induction of EMT in breast cancer. Derksen et al. (2006) confirmed that E-cadherin loss was responsible for anoikis-resistance and increased angiogenesis associated with metastasis in breast cancer, therefore suggested that E- cadherin is essential for tumour initiation and progression. Onder et al. (2008) also found that a truncated form of E-cadherin, lacking external domain of the protein, was sufficient to induce EMT in mammary epithelial cells. These findings were similar to Liu et al. (2006b), in which they noticed two distinct regulatory functions of E- cadherin; mediating an increase in proliferation at low density and inhibiting proliferation at higher densities.
In breast cancer, partial or total loss of E-cadherin is linked to increased invasiveness (Siitonen et al., 1996), metastasis (Oka et al., 1993; Hunt et al., 1997) and poor prognosis (Heimann et al., 2000). Qureshi et al. (2006) found that loss of E- cadherin expression correlated with prognosis and inversely with increased risk of metastasis in invasive ductal carcinoma. Rakha et al. (2005) suggested that loss of E- cadherin in non-lobular breast cancers assists in identifying patients with the worst prognosis. Similarly, Mahler-Araujo et al. (2008) found that poor prognosis non-lobular breast carcinomas that have triple-negative and metaplastic characteristics were associated with reduced E-cadherin expression.
Emerging evidence in the literature suggests that E-cadherin expression is essential in subsequent steps in EMT and cancer metastasis, but E-cadherin loss alone was not sufficient to start the invasion cascade in breast cancer cells (Onder et al., 2008). Knocking down the expression of E-cadherin in normal-like breast cancer cell line MCF10A was insufficient to activate EMT nor increased the aggressiveness of this cell line (Chen et al., 2014). Likewise, restoring E-cadherin expression in the E- cadherin knocked down cells did not restore the full epithelial morphology of MCF10A showing that E-cadherin expression alone was not enough to reverse mesenchymal transition (Li and Mattingly, 2008). Thus, loss of E-cadherin alone does not initiate invasion, even if it resulted in mesenchymal phenotypic change. Similarly, the
restoration of E-cadherin alone did not reverse the mesenchymal morphology of cells. Therefore, suggesting that the loss of E-cadherin is transient and can be reversed.
Re-expression of E-cadherin is essential and parenchyma-tumour cell-cell contact is necessary to induce MET to establish metastases in distant site. Thus, the loss of E-cadherin is transient, and re-expression of the protein occurs by mesenchymal- epithelial transition (MET) after establishing metastasis. Increased E-cadherin expression in metastatic ovarian cancer and high-grade glioma was associated with an increase in tumour invasiveness and poorer prognosis (Sundfeldt, 2003; Reddy et al., 2005; Lewis-Tuffin et al., 2010). Furthermore, Chao et al. (2010) found that E-cadherin expression increased at the metastatic site in 60% of cases of breast cancer compared to the original tumour. Also, the MDA-MB-231 (TN and basal-like) breast cancer cell line re-expresses E-cadherin when co-cultured with liver cells and when injected into mouse tail. Therefore, they concluded that E-cadherin expression was essential to complete tumour metastasis and that loss of E-cadherin in tumour cells is reversible (Chao et al., 2010). Normal parenchymal cells from normal breast tissue was able to restore E- cadherin in the MDA-MB-231 cell line, however, culturing the same cells in the normal extracellular matrix or normal parenchyma-conditioned medium was not sufficient to change the mesenchymal phenotype to an epithelial one (Chao et al., 2010).
In conclusion, loss of E-cadherin is the first step of EMT, yet this loss was not permanent, and re-expression was essential to promote secondary tumour establishment in breast cancer. Furthermore, acquisition of additional invasive properties is necessary for breast cancer progression in addition to the loss of E-cadherin (Onder et al., 2008; Kumar et al., 2011). It was suggested that breast cancer cell metastasis requires a reduction of E-cadherin and increased expression of other EMT-associated proteins (Nieman et al., 1999; Hazan et al., 2000). Therefore, assessing the expression of E- cadherin alone does not reflect the status of EMT or invasiveness of breast cancer because the loss or changes in expression is not a permanent situation. So, it is essential to include additional markers in the confirmation of the EMT status in the assessment of the changes of this process in breast cancer.
1.3.1.2 Increased expression of mesenchymal markers is associated with metastasis and increased cellular motility
Vimentin is a type III intermediate filament that is expressed in normal cells of mesenchymal origin (Gilles et al., 2003; Herrmann and Aebi, 2004; Liu et al., 2015). It is an essential filament during the developmental stages of the mammary gland and for
maintenance of tissue integrity (Liu et al., 2015). It is highly organised and is involved in dynamic cellular processes. It is also associated with intracellular organelles and cell adhesion molecules (Kokkinos et al., 2007). Thus, vimentin protein maintains cellular structure and function (Kokkinos et al., 2007). Increased expression of this marker has been documented in several physiological processes including migration of epithelial cells in embryogenesis and organogenesis, and also during wound healing (Gilles et al., 2003; Kokkinos et al., 2007; Lundgren et al., 2009).
In the human breast, myoepithelial cells express vimentin (Qu et al., 2015). It is also expressed in fibroblasts, stromal cells, leukocytes, and it is low in epithelial cells (Kokkinos et al., 2007). The vimentin promoter is targeted by the b-catenin/TCF transcription factor in mammary cells (Gilles et al., 2003). Thus, the activation of EMT by the Wnt/b-catenin pathway results in increased mammary epithelial cell migration, caused by physiological and pathological processes (Gilles et al., 2003; Kokkinos et al., 2007).
Derksen et al. (2006) found that most adenocarcinomas and solid carcinomas that express an epithelial phenotype lacked expression of vimentin. Meanwhile carcinomas of metaplastic and biphasic histology express a high level of vimentin and lack expression of E-cadherin.
Increased expression of vimentin was associated with increased EMT and linked to increased metastatic potential in breast cancer. Cell lines that show increased vimentin expression had increased fibroblastic morphology, with increased motility and migration through a Matrigel® matrix; whereas cells that have low expression of
vimentin, had more epithelial-like morphology and decreased motility (Sommers et al., 1991). Loss of epithelial cytokeratin alongside an increase in vimentin expression was associated with lymph node metastasis, high tumour grade, poor prognosis and reduced disease-free survival (Chen et al., 2008; Vora et al., 2009). Hemalatha et al. (2013) found that increased expression of vimentin was associated with higher breast cancer tumour grade and with increased proliferative marker expression.
Association of this marker with the different subtypes of breast cancer has been reported. Increased expression of vimentin is associated with high-grade ductal carcinomas, but not with lobular carcinoma (Hemalatha et al., 2013; Liu et al., 2015). The expression of vimentin was also associated with low ER and PR breast cancers, as well as with increased invasiveness of the basement membrane (Kokkinos et al., 2007; Sarrio et al., 2008; Hemalatha et al., 2013; Liu et al., 2015). Moreover, it was reported
that vimentin overexpression is associated with TNBC and BLBC (Sarrio et al., 2008; Kasper et al., 2009; Ricardo et al., 2011). Studies also concluded that increased vimentin expression is associated with HER2 positivity, metastasis and advanced disease stage (Ribeiro et al., 2013; Tanaka et al., 2016). (Hemalatha et al., 2013). Furthermore, it was reported in several studies that loss of E-cadherin expression in highly invasive breast cancer cell lines is associated with increased expression of vimentin (Gilles et al., 2003; Chen et al., 2008).
Increased vimentin expression was seen at the invasive front of breast cancer along with increased b-catenin expression in the cytoplasm and nucleus and the loss of E-cadherin localisation at cell-cell adhesions (Gilles et al., 2003; Sarrio et al., 2008). Several studies reported that b-catenin functions in EMT by regulating the expression level of vimentin (Mestdagt et al., 2006; Wang et al., 2015). Liu et al. (2015) found that vimentin increases the aggressiveness of cancer cells by increasing their tolerance to extracellular stress by maintaining mechanical homeostasis and reorganising the cellular cytoskeleton.
High expression of vimentin has been seen in circulating tumour cells of patients with breast cancer, indicating that it was associated with increased early metastasis (Kallergi et al., 2011). Dwedar et al. (2016) found that hypomethylation of the vimentin gene was frequently associated with TN breast cancer. Yamashita et al. (2013) also found that vimentin expression was associated with poorer prognosis in TN breast cancer cases and suggested its use as a prognostic marker.
However, no association was found between vimentin expression and tumour size, lymph node involvement and patient survival (Hemalatha et al., 2013). One of the reasons for this apparent contradiction in reporting is vimentin expression within the stem cells of the breast. It was concluded that the expression of vimentin in breast tissue is associated with cells that have bilinear origin or differentiation potential (epithelial and myoepithelial) and that it was not associated with the process of EMT (Hemalatha
et al., 2013). Many studies indicated that vimentin expression is associated with stem
cell-like properties in breast cancer cells (Manuel Iglesias et al., 2013; Liu et al., 2014; Qu et al., 2015).
In conclusion, the assessment of more than one marker is essential for the estimation of the epithelial/mesenchymal status of breast cancer cells. Furthermore, expression scoring, and subcellular localisation of the markers must also be considered.
In addition to the decrease in E-cadherin and increase of the free pool of b- catenin, increased vimentin expression is indicative for active EMT in human cancer (Gilles et al., 2003; Sarrio et al., 2008). Increased expression of vimentin was associated with several pathological conditions including human tumours (Conacci- Sorrell et al., 2003; Ha et al., 2013).