Principles of stem cell biology and cancer: future applications and therapeutics. Edited by T. Regad, T. J. Sayers and R. C. Rees. John Wiley & Sons (2015)
Part II. Cancer stem cells
Several recent reviews have provided comprehensive details of the molecular pathways regulating EMT and its counterpart, MET (Lee et al., 2006; Kalluri and Weinberg, 2009; De Craene and Berx, 2013; Lamouille et al., 2013, 2014). The purpose of this section is to provide an overview of the major components involved in the activation of the EMT programme and to provide a framework for further discussions relating to the role of EMT in metastasis. Integration of multiple molecular signalling pathways and regulators is required in order to activate the programme and to mediate the phenotypic and functional changes observed in EMT. These components can be placed into one of three broad categories, based on their functional attributes (Tsai and Yang, 2013): the extracellular microenvironmental stimuli that activate EMT signalling pathways are known as ‘EMT inducers’, the transcription factors that orchestrate and control EMT are called the ‘core regulators’ and the molecules that execute the programme are termed the ‘effectors’ (Tsai and Yang, 2013).
6.2.1. EMT inducers
The induction in epithelial cancer cells of the EMT programme occurs, in large part, through extracellular stimuli present within the tumour microenvironment, including cellular constituents such as CAFs and myeloid-derived suppressor cells (MDSCs), soluble mediators such as growth factors and cytokines and environmental conditions such as hypoxia and acidosis (Gao et al., 2012; Hanahan and Coussens, 2012; Kang and Pantel, 2013; Tam and Weinberg, 2013; Pattabiraman and Weinberg, 2014) (Figure 6.1). Importantly, the heterogeneity inherent to the tumour microenvironment defines the context and tissue-type and cell-type dependence of EMT inducers (Tam and Weinberg, 2013; Tsai and Yang, 2013). Thus, the specific inducers of the EMT programme in cancer are dependent on tissue context and may vary from tumour to tumour, but all induce a common set of signal pathways that make up the core EMT programme. These inducers activate EMT through the coordinated regulation of several developmental signalling pathways, including TGF-β, Wnt, Notch and NF-kB signalling cascades (Figure 6.1). The prototypic inducer of EMT, TGF-β, initiates the programme through stimulation of the TGF-β pathway, in association with the Wnt/β-catenin/LEF-1 and Ras kinase pathways (Nieto, 2013; Tam and Weinberg, 2013). Signalling through the TGF-β pathway results in the formation of a transcription factor complex that migrates to the nucleus and induces a transcription programme that mediates acquisition of mesenchymal traits and suppression of epithelial properties (Tsai and Yang, 2013). Similarly, inflammatory cytokines such as TNF-α can activate EMT through the NF-kB pathway. Hypoxia, through the stabilization and activation of HIF-1a, can also activate and promote EMT (see Section 6.6) (Marie-Egyptienne et al., 2013; Tsai and Yang, 2013). Induction of EMT through cooperative regulation of multiple signalling pathways culminates in the expression and activation of the core transcriptional regulators of EMT.
Figure 6.1. Core components of the activation of the EMT programme in cancer cells. The cells, soluble mediators and microenvironmental conditions that make up the heterogeneous tumour microenvironment serve to induce EMT through the coordinated activation and regulation of multiple intracellular signalling pathways. The activation of these pathways results in the expression and stimulation of several core transcriptional regulators. Once activated, these EMT TFs modulate the differential regulation of several effector molecules of EMT, including the downregulation and repression of epithelial junctional proteins, especially E-cadherin, and the upregulation and activation of mesenchymal proteins such as vimentin and SMA.
6.2.2. Core regulators
In epithelial cancers, the induction of EMT by extracellular stimuli and subsequent activation of signalling cascades results in the expression and stimulation of transcription factors that mediate the regulation of a myriad of genes involved in the processes of cell adhesion, mesenchymal differentiation, migration and invasion (Kalluri and Weinberg, 2009; Scheel and Weinberg, 2012; Nieto, 2013; Tsai and Yang, 2013). The core transcriptional regulators of EMT, known collectively as EMT transcription factors (EMT TFs), incorporate three separate protein families: the zinc-finger family transcription factors Snail1 and Snail2, the E-box-binding homoeobox-family transcription factors Zeb1 and Zeb2 and the basic helix-loop-helix (bHLH)-family transcription factors Twist1 and Twist2 (Kalluri and Weinberg, 2009; Tsai and Yang, 2013) (Figure 6.1). Importantly, these transcription factors may induce EMT alone or in cooperation with one another other. All of them serve to downregulate cell junctional protein expression, especially E-cadherin expression. Snail and Zeb also suppress claudins and ZO-1 junctional proteins, while Twist activates processes critical for cell invasion, demonstrating that these transcription factors are robust mediators of EMT, directing multiple components of the programme (Scheel and Weinberg, 2012; Tam and Weinberg, 2013). These core transcriptional regulators function to modulate the expression of the effector molecules involved in EMT.
The key effector molecules of EMT, including E-cadherin, N-cadherin, vimentin, cytokeratins and SMA, are involved in the conversion from an epithelial phenotype to a mesenchymal phenotype, as well as the promotion of cell migration and invasion (Tsai and Yang, 2013) (Figure 6.1). Specifically, epithelial cancer cells must downregulate junctional proteins, the most critical of which is E-cadherin, the protein regarded as the seminal gatekeeper of the epithelial state (Nieto, 2013; Tam and Weinberg, 2013). In fact, E-cadherin is subject to multiple levels of regulation, including transcriptional repression, promoter methylation and protein degradation. In conjunction with repression of E-cadherin expression, cells undergoing EMT increase their levels of the intermediate filament protein vimentin, as well as SMA. The differential regulation of these key genes allows for the phenotypic conversion of epithelial cells to mesenchymal cells and the acquisition of the migratory and invasive behaviours critical for metastasis.