By contrast with developmental epithelial-mesenchymal transition (EMT), where epithelial characteristics undergo transformation to a mesenchymal-like phenotype in a coordinated fashion, oncogenic EMT occurs in the context of unpredictable genetic changes present in the tumour cells, as well as an abnormal tumour microenvironment. Therefore, a partial form of EMT has been proposed as variably participating in the establishment of invasive phenotype in different types of breast carcinoma, in keeping with their morphological and phenotypical diversity. A complex network of signalling pathways and transcription factors appears responding to various growth factors and cytokines released by stromal and neoplastic elements, endowing the system with abundant regulatory opportunities. The process of EMT is largely elusive in histopathological preparations, prompting doubts regarding its significance in tumour progression. This might be related to the presumed focal occurrence of EMT in the majority of tumours. Detailed topological studies might facilitate understanding of the orchestration of events taking place in vivo. Even more importantly, clinical correlations can be endeavoured and, in parallel with advancement in molecular pathology, a contribution to taxonomy refinement can be envisaged.
- Breast Cancer
- Breast Pathology
- Cancer Research
- Tumour Biology
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A prerequisite for cancer metastasis to occur is that at least a subpopulation of malignant cells should acquire the ability to migrate and invade focally.1 At least in some cases, in order to achieve this ability, carcinoma cells have to partially or completely lose many of their epithelial characteristics and undergo a transformation to a mesenchymal-like phenotype.2 In its fully developed form, this epithelial-mesenchymal transition (EMT) is characterised by architectural changes secondary to loss of cell-cell adhesion structures and modulation of polarity, acquisition of spindle shape, changes of differentiation markers, including downregulation of cell-cell junction proteins and reversion of intermediate filaments from keratin to vimentin, and functional changes associated with increased migratory and invasive capacity.2–4 In developmental EMT, such changes proceed through a coordinated and specific series of events. This is exemplified by the morphogenetic transformations occurring during gastrulation, when mesoderm formation arises at specific sites of primitive ectoderm, where the epithelial cells lose their tight cell-cell adhesions, undergo mesenchymal differentiation and start migrating.3
The role of E-cadherin, β-catenin and Wnt signalling
At cell-cell adherens junctions of epithelial cells, E-cadherin molecules interact in a homophilic manner using their extracellular domains. With their intracellular domains, and through associated catenins, they interact with cytoskeletal proteins, conferring stability on cell-cell adherens junctions.5 E-cadherin has emerged as one of the caretakers of the epithelial phenotype.2 Mutations in the CDH1 gene encoding for E-cadherin are common in invasive lobular carcinomas.6 Whereas lack of E-cadherin expression is one of the defining features of invasive lobular carcinoma, other types of breast cancer show variable downregulation of E-cadherin expression7 ,8 as a result of epigenetic transcriptional events. In addition to regulation by transcriptional factors (see below), transcriptional downregulation can occur by hypermethylation and silencing.9 Reduced E-cadherin expression in breast cancer cells is often associated with inappropriate expression of N-cadherin which is normally expressed by mesenchymal cells.10 Variability in the immunohistochemical N-cadherin staining was seen in a proportion of cells in approximately one-third of invasive ductal carcinomas.11 Another direct consequence of E-cadherin downregulation or loss is the release of β-catenin into the cytosol and potentially into the nucleus,3 where it can bind the LEF/TCF transcription factor. Experiments with human breast cancer cells have shown that one of the targets of the β-catenin/TCF pathway is the vimentin promoter, suggesting a role for β-catenin in the regulation of EMT.12 In addition, β-catenin has been implicated as an effector of Wnt signalling. Wnt proteins are bound tightly to the extracellular matrix, and have been shown to be overexpressed together with Frizzled receptors in human breast cancer cell lines, implying potential autocrine/paracrine stimulation of the Wnt/β-catenin pathway.13 Immunohistochemical analysis in breast cancer TMAs suggested that early activation of this pathway is partially induced by HER2.14
Mechanisms of EMT induction
Following-up initial in vitro observations showing the effect of medium from cultures of human embryo fibroblasts in the morphology and behaviour of epithelial cells,15 there is now strong evidence that stromal factors and their receptors participate in the induction of EMT, and their signalling pathways cross-talk extensively with one another and with oncogenes. Transcription factors also control key steps in EMT. The relevance of such pathways to breast cancer will be discussed, and the potential and limitations of histopathological material in the study of EMT will be contemplated.
Tyrosine kinase receptors-associated pathways
Receptor tyrosine kinases are classified into 20 subfamilies, including EGFR, insulin receptor, PDGFR, VEGFR, Met and FGFR.16 ,17 Binding of growth factors produced by stromal elements induces receptor dimerisation and subsequent transautophosphorylation,18 facilitating binding of several proteins, some of which possess intrinsic enzymatic activity, whereas others participate in ras-mediated signal transduction cascades.19 Activating ras mutations are a common and early event in colorectal carcinogenesis. It is possible that oncogenic activation of its signalling system not only provides a continuous growth stimulus for these cancers, but also potentially increases the propensity for EMT induction.20 Activation of Ras is crucial for protection against anoikis, triggered when cells lose attachment to solid substrates, thus enabling cells to grow in an anchorage-independent fashion.21 All pathways downstream of Ras (PI3K, Raf, Ral-GEF) have been shown to induce EMT.22 Squamous cell carcinoma cells expressing constitutively in vitro an active mutant form of the serine/threonine kinase Akt, downstream of PI3K, displayed features typical of EMT.23 In an experimental animal model, Ras activation alone failed to initiate prostate cancer development, but accelerated progression induced by phosphatase and tensin homolog (PTEN) (a phosphatase that reverses the actions of PI3K) loss and concomitant EMT.24 A hyperactive Raf/MAPK pathway was required for EMT in mammary epithelial cells in vitro.25 Activation of the PI3K and MAPK pathways in breast cancer was evaluated immunohistochemically, using antiphospho-Akt (pAkt) and antiphospho-ERK1/2 antibodies.26 ,27 Non-neoplastic tissue showed minimal or no staining, whereas in tumour cells, variable nuclear and cytoplasmic positivity was noted, with the proportion of phospho-Akt-positive cells ranging from 0% to 85%.26 Interestingly, staining was particularly heterogeneous at the invasive front. Consistent with the finding that activation of the erbB family of receptor tyrosine kinases can initiate and sustain aspects of EMT,28 expression of pAkt (but not pERK1/2) was associated with HER2 overexpression, decreased overall survival in a cohort of lymph node-negative breast cancers, and decreased apoptosis.27 The third Ras-mediated signalling pathway (through Ral-GEF) exerts negative regulatory effect on Rho family GTPases Cdc42 and Rac (discussed below). Regarding elements upstream from this, in vitro studies showed that silencing Ral decreased invasiveness in Matrigel with associated decreased expression of EMT markers.29
The kinase complex FAK-Src, which participates in the signalling cascade initiated by integrin binding to extracellular matrix, also transduces signals from tyrosine kinase receptors.30 In vitro and immunohistochemical analysis of head and neck tumours showed expression of p-Src to be positively correlated with vimentin and negatively correlated with E-cadherin expression.31 Several studies highlight the role of Src in aspects of EMT in breast cancer.32 In hepatocytes, FAK is required for the EMT induced by TGFβ.33 Features of EMT were correlated with increased expression of FAK in breast cancer cells, and were inhibited by micro-RNA 7.34
TGFβ-induced signalling pathways
There is evidence that at the invasive edges of carcinomas, stromal factors facilitate induction of malignant cells to undergo EMT.35 ,36 Among these factors, TGFβ, a cytokine produced by mesenchymal stromal and inflammatory cells,37 is probably the most extensively studied. In view of its established role as inhibitor of cell growth in normal cells,38 via its ability to target the genes encoding the two cyclin-dependent kinase (CDK) inhibitors p15INK4B and p21Cip1 and shut-down expression of their repressor myc,39 TGFβ was first described as tumour suppressor.40 Paradoxically, in cancer, deviations in the TGFβ signalling system elicit resistance to its ability to suppress proliferation with concomitant acquisition of properties facilitating induction of EMT.41 An elegant demonstration of the role of TGFβ as promoter of invasion and metastasis was provided by a histopathological study on breast cancers published well before the detailed account of TGFβ dichotomous behaviour. Extracellular TGFβ derived from tumour cells was significantly increased at the advancing edges of breast carcinomas and lymph node deposits.42
TGFβ receptor is a serine/threonine kinase that signals by phosphorylating and activating Smad2 and Smad3 transcription factors, which subsequently form complexes with Smad4, and translocate en bloc to the nucleus (‘canonical’ or Smad2/3-dependent TGFβ pathway).43 Nuclear expression of activated pSmad2 by malignant cells in breast carcinomas was inversely correlated with histological grade and lymph node status44 and, in the same context, absence of Smad2 appears to define a small but particularly aggressive subset of breast cancers.45 These histopathological studies are intriguingly paralleled with ongoing in vitro and in vivo data revealing the contrasting functions mediated by Smad2 and Smad3.43
Alternatively to ‘canonical’ signalling pathway, TGFβ can also activate Smad2/3-independent ‘non-canonical’ effectors shared with other EMT-related pathways, including receptor tyrosine kinase-associated signalling systems (see above) and Rho family GTPases (see below).43 Activation of these pathways has been associated with TGFβ induction of EMT and promotion of tumour cell invasion and metastasis. For example, exogenous TGFβ administration in mammary epithelial cells, in vitro, induced activation of the PI3K-Akt signalling pathway with EMT-type phenotypic and morphological changes, including delocalisation of E-cadherin and acquisition of spindle shape.46 Similarly, cDNA microarray analysis in mammary epithelial cells treated in vitro with TGFβ, showed increased expression of effectors of the Ras/Raf/MAP kinase pathway and acquisition of EMT features.47 A combined in vitro/in vivo model of carcinogenesis provided strong evidence that EMT requires TGFβ receptor and oncogenic Ras signalling.25 Ras and Raf mutations were found in four of 20 basal-like, but none of 30 HER2-enriched and luminal breast cancer cell lines. In addition, Ras-associated signalling pathway was specifically activated in the basal-like cell lines.48 These findings can be interpreted as indicating that, similar to colorectal cancer, basal-like breast cancer might have increased susceptibility for EMT49 as a consequence of activation of Ras signalling. The TGFβ/Par6-associated non-canonical pathway has been extensively studied and implicated in linking TGFβ signalling, polarity and tight junction regulation.50
The receptor tyrosine kinase/Ras-associated PI3K signalling pathway induces activation of guanine nucleotide exchange factors (GEFs) that are responsible for the activation of the Rho family small GTPases Rho, Rac and Cdc42.21 Activated Rho proteins regulate cytoskeletal organisation and are thought to be important in EMT.51 Cdc42 stimulates the production of highly branched actin filaments, and participates on extension of filopodia, spikelike structures thought to sense signals and enable movement,51 and invadopodia, regions of proteolytic matrix degradation by matrix metalloproteinases.52 Both filopodia and invadopodia have been implicated as mediators of EMT-induced metastasis.53In vitro, a basal-type breast cancer cell line exhibited high levels of invadopodia activity.54 Cdc42, RhoA and Rac1 were overexpressed in breast cancer, and higher grade was correlated with significantly increased expression of Rho-like proteins.55 However, different studies have indicated contradictory roles for the Rho proteins51 and, despite their closely related sequence identity, RhoA and RhoC appeared exerting different effects on cell morphology, migration and invasion.56 An in vitro study using breast carcinoma cells revealed distinct and inverse roles for RhoA and RhoC, with RhoA inhibiting invasion and RhoC stimulating invasion.57 Considering the above mentioned increased expression, not only of RhoC, but also RhoA in invasive breast carcinoma,55 it was speculated that RhoA may contribute in other ways to breast cancer progression, for instance, by promoting EMT. A correlation between the invasive phenotype, with increased focal adhesion density and lamellipodia formation, increased Rac activity and increased metastatic efficiency, was shown in a human breast cancer cell line.58 In addition, the non-canonical TGFβ/Par6 pathway linking TGFβ signalling, polarity and tight junction regulation, appears to act by mediating localised ubiquitination and degradation of Rho with subsequent loss of tight junctions.59
It is intriguing that, unlike the oncogenic constitutive activation of Ras by point mutation occurring frequently in human cancers, mutations in the coding sequence of genes encoding GTPases of Rho family do not occur frequently in breast cancer.60 The mechanisms underlying the ability of these GTPases to modify the invasive and metastatic potential of breast carcinoma have not been fully understood, but a role of upstream elements, including ligation of growth factor receptors, has been speculated.58
Functional loss of E-cadherin, one of the hallmarks of EMT2 by transcriptional repression is mediated by several transcription factors, including zinc finger members of the snail family (SNA1 and SNA2 or slug), and of the ZEB family (ZEB1 and ZEB2). Basic helix-loop-helix factors (bHLH: E47 and Twist) are also transcriptional repressors of E-cadherin.61 Snail genes are expressed in all EMT processes. Signalling involved in EMT, including tyrosine kinase receptor-associated, and serine/threonine kinase receptor-associated pathways and pathways triggered by Notch and Wnt, have also been shown to induce Snail genes.62 In addition to extensive cross-talk between some of these signalling pathways, additional signals have been implicated in CDH1 transcription. For instance, signalling through the oestrogen receptor downregulates snail expression in breast cancer cells.63 This regulatory pathway predicts that loss of oestrogen receptor would be associated with increased snail expression, in keeping with the increased slug mRNA levels in basal-like breast carcinomas,64 a subtype that has been shown to have reduced E-cadherin expression.65 Slug expression is negatively correlated with E-cadherin in vitro.66 An in vivo model of HER-2-induced breast cancer demonstrated snail upregulation in recurrent tumours that displayed features of EMT, including prominent E-cadherin reduction and spindle-shaped cell morphology.67 However, qRT-PCR in breast tumour samples showed positive correlation between E-cadherin, and both slug and snail, and immunohistochemistry revealed unequivocal colocalisation of nuclear snail and membranous E-cadherin in malignant cells.68 Furthermore, qRT-PCR showed reduced snail expression in patients with local recurrences.69 Although there is a remarkable discrepancy between these findings, some observations in the topology of tumour samples appeared to agree with the concept of downregulation of E-cadherin gene in breast carcinomas by transcription factors. Non-invasive tumours expressed significantly less snail and more E-cadherin than invasive tumours, and levels of slug or snail expression were significantly higher in invasive ductal carcinomas associated with lymph node metastasis, suggestive of a role for snail in tumour progression.68 In addition, a more recent immunohistochemical study showed nuclear accumulation of slug in approximately one-third of invasive ductal carcinomas. Positive nuclear staining was associated with loss of E-cadherin membranous staining.7 Highlighting once more the concept of local occurrence of EMT, a small set of cervical squamous cell carcinomas and colon carcinomas showed snail reactivity at the edge of invading tumour islands, close to stromal cells also expressing snail.70
Expression of Twist mRNA in human breast tumour samples was increased in 70% of invasive lobular carcinomas but only 32% of invasive ductal carcinoma,71 consistent with the reverse E-cadherin expression. Although the majority of invasive ductal carcinomas in this study showed no increased Twist levels, one-third of them did. An interesting finding in this study was that Twist induced in vitro the expression of EMT-related markers like fibronectin and N-cadherin, independent of E-cadherin expression.71 Immunohistochemically, normal breast epithelium showed practically no Twist expression, whereas ductal carcinoma in situ (DCIS) and invasive ductal carcinoma, especially high grade, were positive.72 Using qRT-PCR, levels of Twist transcripts were positively correlated with lymph node involvement in breast tissue samples from a mixed group of invasive carcinomas.69
The concept of epithelial plasticity/partial EMT
By contrast with full-blown developmental EMT, oncogenic EMT occurs in the context of unpredictable genetic changes present in the tumour cells, as well as an abnormal tumour microenvironment,73 and not all steps described in developmental EMT are necessary for the invasive phenotype to be established. As remarked in a recent review, the final result of EMT in vivo is not usually a fibroblast but a partially dedifferentiated cell,4 with metaplastic carcinoma being occasionally an exception to this generalisation. In view of the uncertainty on the proportional contribution of EMT (transdifferentiation) and faulty differentiation (dedifferentiation) process to the morphology and phenotype of a given tumour, the term ‘EMT-like’ has been suggested. Taking into account the state of cell polarisation, state of cell cohesiveness and intermediate filament protein expression, a system comprising four EMT-like phenotypes has been proposed.74 In the same context, the terms ‘epithelial-mesenchymal plasticity’75 and ‘incomplete EMT’76 have been advocated, underlining the continuum from epithelial cells arranged in solid sheets to isolated spindle-shaped cells.73
The emergence of the concept of partial EMT, a process leading to an intermediate phenotype, where some characteristics of epithelium are retained, but features of mesenchymal cells also appear,4 does not come as a surprise to breast pathologists. Invasive breast carcinoma cells are exceedingly diverse morphologically. Their morphology and architectural arrangement ranges from the almost identical to benign luminal epithelial cells of invasive tubular carcinoma to the undifferentiated mesenchymal-like cells of metaplastic carcinoma. Interestingly, polarity of neoplastic cells in Grade I invasive ductal carcinomas did not correlate with survival, indicating that loss of epithelial phenotype and cell polarity is by no means necessary for invasion and metastasis.77 The hypothesis that the contribution of EMT in cancer progression depends on the tumour type4 seems plausible.
Is EMT really happening?
There might be some merit in the doubt arisen relating to the fundamental principle that EMT occurs in real cancers.78 Besides concerns about the reliability of in vitro experiments as models for in vivo phenomena, and the absence of obvious intermediate forms in histological sections, a few more points could be added. Although post-EMT cells might not be appreciable as they lack keratin expression and are morphologically similar to normal vimentin-expressing stromal cells,74 it can be assumed that these cells should still retain the nuclear pleomorphism of their pre-EMT counterparts, a fact that is hardly noticed. In addition, spindle-shaped cells are not regularly noted in metastatic sites like, for instance, in routinely examined axillary lymph nodes. However, counterarguments to these points definitely exist. The strongest is based on the evidence suggesting that EMT occurs in a local manner at the invasive front of a tumour, and that such localised EMT is no less significant.73 In this context, mesenchymal cells with atypical-appearing nuclei can occasionally be recognised but are not systematically sought after in routine examination. A fibrous reaction which is sometimes seen in lymph node metastases,79 could be viewed as a potential mesenchymal-epithelial transition site.
In more general terms, the presumed local occurrence of EMT in some breast carcinomas might be related to the heterogeneous nature of neoplastic and microenvironmental elements. Heterogeneity of the neoplastic component refers to features ranging from obvious morphological characteristics (like, eg, in carcinosarcomas) and hormone responsiveness, to subtle differences in the expression of factors participating in EMT-related pathways. Heterogeneity of the microenvironment refers to its potential for cytokine production, cell-extracellular matrix adhesions80 and matrix metalloproteinase production.81
It is clear that tumour biology cannot be simply extrapolated from the properties of individual cells. By contrast with this reductionist approach, it is now well established that cancer is a disease of complex and heterogeneous cellular architectural organisation. In comparison with tissue culture and experimental animal models, pathological material has the presumed disadvantage of a static arena for cancer research. However, it provides the only means where the authentic architectural organisation of human cancer and its microenvironment can be studied. Detailed topological examination and comparison of breast carcinomas with different grades, and at different stages, can provide a dynamic setting where even temporal information can be deducted, and hypotheses for mechanisms involved in tumour progression can be formed. Such a setting appears to be especially relevant for studies attempting to correlate the complex molecular pathways participating in EMT. Even more importantly, clinical correlations can be endeavoured and, in parallel with advancement in molecular pathology, a contribution to taxonomy refinement can be envisaged.
A partial form of epithelial-mesenchymal transition occurs in different types of breast cancer and affects their invasive phenotype.
Histopathological material provides an ideal setting where the authentic architectural organisation of breast cancer and its microenvironment can be studied.
Detailed topological studies of breast carcinomas can facilitate elucidation of the events taking place during epithelial-mesenchymal transition in vivo.
Funding NIHR Biomedical Research Centre, Oxford.
Competing interests None.
Provenance and peer review Not commissioned; internally peer reviewed.
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