Molecular mechanisms of epithelial-mesenchymal transition

Abstract

The transdifferentiation of epithelial cells into motile mesenchymal cells, a process known as epithelial-mesenchymal transition (EMT), is integral in development, wound healing and stem cell behaviour, and contributes pathologically to fibrosis and cancer progression. This switch in cell differentiation and behaviour is mediated by key transcription factors, including SNAIL, zinc-finger E-box-binding (ZEB) and basic helix-loop-helix transcription factors, the functions of which are finely regulated at the transcriptional, translational and post-translational levels. The reprogramming of gene expression during EMT, as well as non-transcriptional changes, are initiated and controlled by signalling pathways that respond to extracellular cues. Among these, transforming growth factor-β (TGFβ) family signalling has a predominant role; however, the convergence of signalling pathways is essential for EMT.

Figure 1. Cellular events during EMT

a | The first steps of epithelial–mesenchymal transition (EMT) are the disassembly of epithelial cell–cell contacts — that is, tight junctions, adherens junctions, desmosomes and gap junctions — and the loss of cell polarity through the disruption of the Crumbs, partitioning defective (PAR) and Scribble (SCRIB) polarity complexes. The expression of epithelial genes is repressed, concomitantly with the activation of mesenchymal gene expression. b | Next, the epithelial actin architecture reorganizes, and cells acquire motility and invasive capacities by forming lamellipodia, filopodia and invadopodia, and by expressing matrix metalloproteinases (MMPs) that can degrade extracellular matrix (ECM) proteins. The process of mesenchymal–epithelial transition (MET) enables the cells that have undergone EMT to revert to the epithelial state. Three types of EMT can be discerned depending on the physiological tissue context. Type 1 EMT occurs in embryogenesis and organ development, type 2 EMT is important for tissue regeneration and organ fibrosis^,, and type 3 EMT is associated with cancer progression and cancer stem cell properties. MET also contributes to development (for example, kidney development) and the generation of metastatic carcinomas^,. Consecutive rounds of EMT and MET occur during development^,. In primary EMT, ectodermal or epithelial cells without prior history of EMT differentiate into mesenchymal cells. In secondary EMT, cells that have already undergone EMT and reverted to the epithelial state initiate a new EMT process^,. Similarly, following dissemination, cancer cells can revert through MET to an epithelial state, leading to the formation of secondary carcinomas with phenotypes similar to the primary tumour. aPKC, atypical protein kinase C; DLG, discs large; LGL, lethal giant larvae; N-cadherin, neural cadherin; PALS1, protein associated with Lin-7 1; PATJ, PALS1-associated tight-junction protein.

Figure 2. Roles and regulation of major EMT transcription factors

Epithelial–mesenchymal transition (EMT) is driven by SNAIL, zinc-finger E-box-binding (ZEB) and basic helix–loop–helix (bHLH) transcription factors that repress epithelial marker genes and activate genes associated with the mesenchymal phenotype. Post-translational modifications regulate their activities, subcellular localization and stability. a | Glycogen synthase kinase-3β (GSK3β) phosphorylates (P) SNAIL1 at two motifs; phosphorylation of the first motif facilitates the nuclear export of SNAIL1, and phosphorylation of the second motif enables the ubiquitin (Ub)-mediated degradation of SNAIL1. Phosphorylation of SNAIL1 by protein kinase D1 (PKD1) also leads to its nuclear export. Conversely, phosphorylation of SNAIL1 by p21 activated kinase 1 (PAK1) or large tumour suppressor 2 (LATS2), or dephosphorylation of SNAIL1 by small C-terminal domain phosphatase1 (SCP1) promotes the nuclear retention of SNAIL1 and enhances its activity. SNAIL2 is degraded as a result of its p53-mediated recruitment to the p53–mouse double minute 2 (MDM2) complex. b | TWIST is phosphorylated by the MAPK p38, JUN N-terminal kinase (JNK) and ERK, which protects it from degradation, and thus promotes its nuclear import and functions. c | ZEB2 is sumoylated (Sumo) by Polycomb repressive complex 2 (PRC2) and subsequently exported from the nucleus, which reduces its activity as a transcription factor. E-cadherin, epithelial cadherin; ID, inhibitor of differentiation; MMP, matrix metalloproteinase; N-cadherin, neural cadherin; PALS1, protein associated with Lin-7 1; PATJ, PALS1-associated tight-junction protein; SPARC, secreted protein acidic and rich in Cys; ZO1, zonula occludens 1.

Figure 3. Molecular mechanisms of TGFβ-induced EMT

The initiation of, and progression through, epithelial-mesenchymal transition (EMT) are regulated at the transcriptional, post-transcriptional, translational and post-translational levels. Transforming growth factor-β (TGFβ) induces EMT by acting at several of these levels and through SMAD-mediated and non-SMAD signalling. TGFβ signals through a tetrameric complex of type I and type II receptors (TβRI and TβRII) to activate SMAD2 and SMAD3, which then combine with SMAD4. The trimeric SMAD complex translocates into the nucleus and cooperates with transcription regulators in the repression or activation of target genes. TGFβ –SMAD signalling activates the expression of EMT transcription factors, and SMAD complexes cooperate with these transcription factors to increase their transcriptional activities. TGFβ also induces the expression of microRNAs (miRNAs) that repress the expression of epithelial proteins, and EMT transcription factors can repress the expression of miRNAs that target mesenchymal components, thus promoting EMT. TGFβ signalling also decreases the expression of the epithelial splicing regulatory proteins (ESRPs), which leads to a differential splicing programme following EMT. TGFβ can also induce non-SMAD signalling pathways that contribute to EMT. It activates PI3K–AKT-mammalian TOR complex 1 (mTORC1) signalling, which increases translation and cell size; active AKT also derepresses the translation of specific mRNAs by phosphorylating heterogeneous nuclear ribonucleoprotein E1 (hnRNPE1). TGFβ also increases cell junction dissolution and induces cytoskeletal changes by regulating RHO-GTPases. TGFβ induces TβRII association with a TβRI-partitioning defective 6 (PAR6) complex at tight junctions, which enables TβRII to phosphorylate PAR6; this results in the recruitment of the E3 ubiquitin ligase SMAD ubiquitylation regulatory factor 1 (SMURF1), RHOA ubiquitylation and degradation, and the loss of tight junctions. TGFβ also induces RHOA activity; this promotes actin reorganization by leading to the activation of diaphanous (DIA1) and also RHO-associated kinase (ROCK), which phosphorylates myosin light chain (MLC) to activate LIM kinase (LIMK) and thus inhibit cofilin. RAC and CDC42 also participate in cytoskeletal changes through p21 activated kinase 1 (PAK1) and direct the formation of lamellipodia and filopodia. 4E–BP1, eukaryotic translation initiation factor 4E–binding protein 1; S6K1, ribosomal S6 kinase 1.

Figure 4. Signalling pathways involved in EMT

Epithelial-mesenchymal transition (EMT) progression is regulated by signalling pathways that can cooperate to induce full EMT responses. In addition to promoting EMT through SMAD proteins, transforming growth factor-β (TGFβ) can activate the PI3K–AKT, ERK MAPK, p38 MAPK and JUN N-terminal kinase (JNK) pathways. TβRI phosphorylates the adaptor protein SRC homology 2 domain-containing-transforming A (SHCA), which creates a docking site for growth factor receptor-bound protein 2 (GRB2) and son of sevenless (SOS) and initiates the RAS-RAF-MEK-ERK MAPK pathway. TGFβ-induced p38 MAPK and JNK activation results from the association of TNF receptor-associated factor 6 (TRAF6) with the TGFβ receptor complex, which activates TGFβ-activated kinase 1 (TAK1) and p38 MAPK and JNK as a result. Several growth factors that act through receptor tyrosine kinases (RTKs), including epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), can induce EMT. The RAS-RAF-MEK-ERK MAPK signalling cascade represents a major pathway that is activated by RTKs in response to growth factors. Once activated ERK1 and ERK2 MAPK can facilitate EMT by increasing the expression of EMT transcription factors and regulators of cell motility and invasion, such as RHO GTPases and the 90 kDa ribosomal protein S6 kinase. Other signalling pathways, such as the WNT, Notch and Hedgehog (HH) pathways, also participate in EMT. WNT signalling promotes EMT by inhibiting glycogen synthase kinase-3β (GSK3β) to stabilize β-catenin, which translocates to the nucleus to engage the transcription factors lymphoid enhancer-binding factor 1 (LEF) and T cell factor (TCF) and promote a gene expression programme that favours EMT. In HH signalling, glioma 1 (GLI1) can induce SNAIL1 expression, and the intracellular domain of Notch can activate SNAIL2 expression; thus HH and Notch signalling promote a decrease in epithelial cadherin (E-cadherin) levels. The cell microenvironment also regulates EMT. During inflammation and in cancer, interleukin-6 (IL-6) can promote EMT through Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3)-induced SNAIL1 expression. Hypoxia in the tumour environment can promote EMT through hypoxia-inducible factor 1α (HIF1α), which activates the expression of TWIST. EMT responses can be increased through crosstalk and cooperation between distinct pathways. For example, RTK- or integrin-induced AKT activation can induce SNAIL expression through nuclear factor-κB (NF-κB) and stabilize SNAIL and β-catenin by inhibiting GSK3β , thus cooperating with WNT signalling. TGFβ signalling can also increase EMT responses initiated by growth factors such as FGF or EGF. ECM, extracellular matrix; FZD, frizzled; ILK, integrin-linked kinase; MKK, MAPK kinase; mTORC2, mammalian TOR complex 2; Notch-IC , intracellular fragment of Notch; PTCH1, patched 1; SHH, sonic HH; SMO, smoothened.