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REVIEWS Regulatory networks defining EMT during cancer initiation and progression Bram De Craene and Geert Berx Abstract | Epithelial to mesenchymal transition (EMT) is essential for driving plasticity during development, but is an unintentional behaviour of cells during cancer progression. The EMT-associated reprogramming of cells not only suggests that fundamental changes may occur to several regulatory networks but also that an intimate interplay exists between them. Disturbance of a controlled epithelial balance is triggered by altering several layers of regulation, including the transcriptional and translational machinery, expression of non-coding RNAs, alternative splicing and protein stability. E‑cadherin This protein is a classical cadherin. Cadherin cytoplasmic domains interact with a catenin-based complex, which couples to the actin cytoskeleton and regulates adhesion-dependent signalling. Loss of function may contribute to cancer progression by increasing proliferation, invasion and/or metastasis. Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, 9052 Zwijnaarde, Belgium; and Unit of Molecular and Cellular Oncology, Department for Molecular Biomedical Research, VIB, 9052 Ghent, Belgium. Correspondence to G.B. e‑mail: Geert.Berx@dmbr. VIB-UGent.be doi:10.1038/nrc3447 During epithelial to mesenchymal transition (EMT) epithelial cells are converted to migratory and invasive cells. This process has been considered to be a fundamental event in morphogenesis as it is intimately involved in the generation of tissues and organs during embryogenesis of both vertebrates and invertebrates1. A similar process is recapitulated during wound healing, a classical example of a process in adulthood in which EMT is important. It has now been more than 10 years since EMT was first demonstrated to be closely related to cancer progression2,3 (BOX 1). The ability of the transcription factor SNAI1 to directly repress E-cadherin has been considered dogma since its initial observation4,5, and this conviction has been reinforced over the years. The list of potent EMT-inducing transcription factors (EMT-TFs) has been growing ever since 6, revealing regulatory networks that are often described as signalling pathways converting stimuli to transcriptional reprogramming 7,8. Endogenous expression of EMT-TFs has been found in various tissues and is not necessarily coupled to the dedifferentiation of tumour cells. Therefore, their actual role is broader than the name EMT‑TF suggests. A thorough understanding of their molecular function can increase our knowledge of their specific contribution in the context of cancer. Indeed, EMT‑TFs have been involved not only in migration and invasion but also in the suppression of senescence and apoptosis, attenuation of cell-cycle progression and resistance to radiotherapy and chemotherapy 1,9,10 (FIG. 1). In addition, expression of SNAIL family members, such as SNAI1 and SNAI2 (also known as SLUG), has been observed in several stem-cell compartments, such as those of melanocytes11, haematopoietic cells12 and the epithelial compartment of the gut 13. SNAIL transcription factors probably actively participate in the maintenance of plasticity during stemcell homeostasis or exploit the plasticity during stem-cell expansion and differentiation, as has also been suggested for mammary-gland development 14,15. The importance of this EMT-related reversible plasticity is clearly emphasized during embryogenesis and cancer progression, in which the reverse process — mesenchymal to epithelial transition (MET) — is important for final developmental cellular differentiation and for clonal outgrowth at metastatic sites8,16. Furthermore, the EMT–MET balance seems to fine-tune cellular destiny, as exemplified by the MET being crucial for the reprogramming of fibroblasts towards induced pluripotent stem (iPS) cells17,18. However, a pertinent role for EMT-TFs in the normal homeostasis of differentiated epithelial cells remains elusive, although they are naturally present in various non-epithelial differentiated cell types, such as haematopoietic cells19, lymphocytes20 and neurons21. Importantly, these nuclear drivers of EMT seem to be involved in both differentiation and dedifferentiation, and this implies that their function is compatible with both growth suppression and stimulation, depending on the context. For an epithelial cell to undergo a full EMT, the cell should be permissive: the mesenchymal end-stage can be considered the ultimate goal and will only be achieved when all necessary component pathways of the network are activated. There is good evidence to support the idea that dedifferentiation is triggered if the balance between different regulatory networks is fundamentally NATURE REVIEWS | CANCER VOLUME 13 | FEBRUARY 2013 | 97 © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS At a glance •Epithelial to mesenchymal transition (EMT) will lead to reversible reprogramming of the cell, which is defined by fundamental changes initiated and maintained by several regulatory circuits. •EMT is well known to be transcriptionally regulated. Several transcription factors have been described as potent enough to drive EMT. The often strong interconnection between these factors forms a solid network that drives tumour progression. •Recent evidence has linked EMT to epigenetic modifications. These are reversible, thus emphasizing that epigenetic modifications may contribute to EMT plasticity, which could allow cancer cells to switch back to the epithelial state on colonization at a secondary site. •Non-coding RNAs, and in particular microRNAs (miRNAs), are master regulators of gene expression in many biological and pathological processes. Several miRNAs are able to influence the cellular phenotype through the suppression of genes that are involved in controlling the epithelial and mesenchymal cell states. Moreover, feedback loops with transcriptional regulators of EMT further define and/or maintain a given cellular state. •Alternative splicing of mRNA precursors leads to the formation of different proteins from the same gene, and this directs distinct physiological functions. EMT-associated alternative splicing events are regulated by several recently identified proteins, adding a new layer to the complex regulation of EMT. •More recent studies have indicated that protein levels of EMT-inducing transcription factors are tightly controlled by additional mechanisms. A first level of control is found at the point of translation initiation and elongation. A complex machinery determines the stability, subcellular localization and functionality of the proteins. Misregulation of translation and post-translational regulation may contribute to EMT in cancer cells. Mesenchymal to epithelial transition (MET). The conversion of non-polarized and motile mesenchymal cells into polarized epithelial cells. MET is typically associated with increased E‑cadherin levels and low cancer cell invasion and metastasis. It is the reverse of epithelial to mesenchymal transition. Vimentin Intermediate filament found in various non-epithelial cells, particularly mesenchymal cells. disturbed; this probably requires the disruption of more than one layer (FIG. 2). Indeed, misexpression of SNAI1 or ZEB2 in transgenic mouse models disturbs homeostasis in some tissues, but these mice are viable and the status of many epithelial compartments is still maintained22,23 (M. N. Tatari and G.B., unpublished observations), hence the cell is not fully permissive and EMT is unable to occur. EMT in epithelial cells is often observed when the expression of an EMT‑TF is combined with an additional acquired oncogenic activity. For example, TWIST1 expression will promote tumour initiation and progression in vivo only when combined with activated RAS24. A recent study by Rhim and colleagues25 further supports the concept of permissiveness, using a model of pancreatic cancer in which yellow fluorescent protein (YFP)-labelled pancreatic epithelial cells have a mutant Kras combined with loss of Trp53. On the one hand, induction of EMT at an early stage is sufficient for the cells (that is, permissive cells) to escape the primary lesions, supporting the possibility of parallel tumour progression (FIG. 1). On the other hand, 42% of the YFP-positive cells in these tumours had undergone EMT, supporting the existence and potency of EMT during primary tumour progression. In fact, all of these in vivo studies provide us with a better understanding of why EMT downstream of an EMT‑TF is much easier to detect in vitro: cell lines are genetically aberrant, resulting in different degrees of plasticity and allowing a range from partial to full EMT to occur. In the past few years it has become clear that EMT is driven by at least four fundamental regulatory networks. These networks are closely connected, and modulation of any of these networks has a profound effect on every other network. The most extensively studied network is undoubtedly the network built around the nuclear factors of the SNAIL, ZEB and TWIST families; this network is supported by various interacting proteins, which results in a strong transcriptional control of EMT. There is growing evidence that three additional layers of regulation solidly support the EMT programme in parallel; these regulatory layers are the expression of small noncoding RNAs, differential splicing, and translational and post-translational control (which affect protein stabilization and localization). In this Review we discuss recent insights into the control of EMT at different molecular levels and their conceptual implications for cancer progression. Transcriptional control of EMT The strength of EMT is primarily dependent on the potency of EMT-inducing transcription factors that are capable of triggering cellular reprogramming. Overall, proteins that are involved in transcriptional control contribute to the best characterized regulatory network during EMT (FIG. 2). After the initial observation of a direct interaction of SNAI1 with the CDH1 promoter (which encodes E‑cadherin)4,5, many other transcriptional repressors such as SNAI226, ZEB1 and ZEB2 (REFS 27,28), E47 (also known as TCF3) 29, Krüppellike factor 8 (KLF8)30 and Brachyury 31 were identified (TABLE 1). The power of these factors relies not only on the direct repression of E‑cadherin, but also on the simultaneous repression of the transcription of several other junctional proteins, including claudins and desmosomes, which facilitates the general dedifferentiation programme32–34. The endogenous presence or the forced expression of these EMT-TFs in cancer cells have been linked with the loss of E‑cadherin and the gain of vimentin, and these observations are the most commonly used to support the importance of EMT in experimental situations. On the basis of these criteria the list of EMT-TFs was further extended to include TWIST1 (REF. 35), forkhead box protein C2 (FOXC2)36, goosecoid37, E2‑2 (also known as TCF4)38, homeobox protein SIX1 (REF. 39) and paired mesoderm homeobox protein 1 (PRRX1)40. These transcription factors seem to trigger EMT without direct binding to the CDH1 promoter. The variety of different factors and associated functions exemplifies the different degrees of dedifferentiation that may be expected, and probably explains the tendency of researchers to speak about partial EMT when dediff erentiation is not fully completed. Remarkably, these EMT-TFs are exclusively associated with promoting the mesenchymal phenotype, supporting the idea that the epithelial phenotype is a default status41. However, it seems that the study of the transcriptional machinery on the epithelial side may have just been lagging behind, as it is now becoming clear that epithelium-specific transcription factors such as grainyhead-like protein 2 homologue (GRHL2) and the ETS-related transcription factors ELF3 and ELF5 are downregulated during EMT and actively drive MET when overexpressed in mesenchymal cells32,42,43. These findings seem to support the alternative concept of a tightly controlled balance 98 | FEBRUARY 2013 | VOLUME 13 www.nature.com/reviews/cancer © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS Polycomb group proteins Proteins first described in Drosophila melanogaster that are required for normal development. They work in multi-protein complexes, called Polycomb repressive complexes, that establish regions of chromatin in which gene expression is repressed. Deamination of trimethylated H3K4 Methylation of lysine 4 (K4) in histone H3 has been linked to active transcription and is removed by LSD1 and the Jumonji C domain-containing proteins by amino oxidation or hydroxylation, respectively. The deamination catalysed by LOXL2 is a novel chemical mechanism for H3K4me3 modification. Transforming growth factor-β (TGFβ). TGFβ family members are potent extracellular factors that can initiate and maintain EMT in various biological systems. Adding TGFβ to epithelial cells in culture is a convenient way to induce EMT in various epithelial cells. between epithelial and mesenchymal status, but they still leave room for partial EMT to occur (FIG. 2). Over the years it has also become clear that many of the EMT-TFs often function in the same pathway or in synergy with each other. Remarkably, although some of them are only mentioned sporadically in the context of EMT, others seem to be common in the majority of the studies, in particular the nuclear factors of the SNAIL, ZEB and TWIST families. Their consistent association with EMT suggests that the collection of master EMT-TFs may be complete, and that most of the other factors may have a facilitating function, or at least they will feed to these master EMT-TFs, finally resulting in EMT. One of the reasons for the high potency, and thus abundance in the literature, of the SNAIL, ZEB and TWIST proteins might be their close interaction with different epigenetic modifiers, thus allowing fundamental genome-wide changes in gene expression. For a long time, epigenetic changes during EMT were primarily studied on the CDH1 promoter. DNA methylation on CpG dinucleotides in regulatory sequences is a typical mechanism for silencing tumour suppressors in cancerous cells. CDH1 promoter methylation has been recognized as part of the programme that results in EMT44,45, and has been associated with SNAI1 expression46. Notably, DNA methyltransferase 1 (DNMT1) potentially modulates CDH1 expression in a methylation-independent context. This could happen by direct interaction with SNAI1 through the amino‑terminal region, which would thereby prevent the association of this transcriptional repressor with the CDH1 promoter 47. Epigenetic modifications also involve histone modifications; in particular, deacetylation and specific Box 1 | EMT and cancer progression: a controversial issue The clinical relevance of epithelial to mesenchymal transition (EMT) during cancer is frequently questioned. This is mainly because evidence of a full EMT phenotype in clinical carcinomas and metastases is generally lacking16. The failure to prove EMT on the molecular level has been countered by the concept that EMT changes are transient and may happen in a minority of cancer cells, which is supported by the clinical observation that single cancer cells or small cell clusters with reduced E‑cadherin levels are present at the invasive front139. The idea that EMT has a role in cancer has also benefited from its link with the loss of E‑cadherin expression, which is a well-established condition for malignant cancer progression. However, E‑cadherin loss is not always related to the induction of EMT: for example, lobular breast cancer has genetic inactivation of CDH1 (which encodes E‑cadherin) but also has epithelial characteristics45. Furthermore, partial or complete loss of E‑cadherin alone is usually not enough to trigger EMT-associated transcriptional reprogramming, and can be the consequence of many EMT-independent cellular changes that do not have a proven role in cancer dissemination140. Evidence for a complete EMT in human cancer is clearly present in carcinosarcomas; most other cancers have partial features of EMT and express both epithelial and mesenchymal markers141. In addition, a transcriptomic EMT signature has been associated with the poor prognosis of patients with basal B or claudin-low breast tumours63,142,143. An active role of EMT during metastasis is also suggested by the finding that the gain of expression of mesenchymal markers in circulating tumour cells of patients with breast cancer predicts poor prognosis more accurately than only considering epithelial cytokeratin markers144. These clinical examples showing the relevance of EMT for human cancer are supported by many xenograft experiments and work with genetically modified mouse cancer models. The ability to sensitively tag cancer cells in experimental models has made it clear that EMT is associated with in vivo epithelial tumour progression and could be involved in the seeding of distant organs before, or in parallel with, tumour formation at the primary site25,145. methylation or demethylation patterns are considered to lead to gene repression (reviewed in REF. 48). It has been shown that SNAI1 induces repressive histone modifications at the CDH1 promoter through the recruitment of many different proteins, such as histone deacetylase 1 (HDAC1) and HDAC2 (REF. 49). Similarly, ZEB1 cooperates with the deacetylase sirtuin 1 (SIRT1) at the CDH1 promoter, leading to the deacetylation of histone H3 and to reduced binding of RNA polymerase II50. Furthermore, various histone modifiers have been linked to SNAI1 activity, such as the demethylase LSD1 (REF. 51) and the methyltransferases EZH2, SUZ12 (REF. 52), SUV39H1 (REF. 53) and G9a (also known as EHMT2)54. ZEB1 probably acts in a similar way to suppress CDH1 by interacting with BRG1, a subunit of the SWI–SNF chromatin complex and a known interaction partner of several histone-modifying enzymes55. EZH2 and Polycomb complex protein BMI1 are Polycomb group proteins, which regulate lineage choices during development and differentiation, and both proteins cooperate with TWIST1 to repress CDH1 transcription56. Remarkably, lysyl oxidase homologue 2 (LOXL2) has a dual role in SNAI1‑driven CDH1 repression: although it was initially shown to be involved in maintaining the post-translational stability of SNAI1 (REF. 57), it also catalyses the deamination of trimethylated H3K4 (REF. 58). In uncontrolled situations such as cancer it seems reasonable to accept that EMT-TFs will interact with many of the available cofactors in the deregulated cell, facilitating EMT. Given the variety of potential modifications at the CDH1 locus, genome-wide epigenetic reprogramming during EMT has recently gained attention, and this has been demonstrated by several genome-wide studies. During hypoxia-induced EMT, histone deacetylase 3 (HDAC3) was identified as a direct transcriptional target of hypoxia-inducible factor 1α (HIF1α). HDAC3 is recruited to the epithelium-specific promoters, such as CDH1 and plakoglobin, where it cooperates with SNAI1 to mediate gene repression59. HDAC3 further interacts with hypoxia-induced WD repeat-containing protein 5 (WDR5), recruiting the histone methyltransferase complex to increase histone-specific methylation, thereby activating mesenchymal genes such as those encoding vimentin and N-cadherin59. Treating cultured mouse hepatocytes with transforming growth factor-β (TGFβ) induces EMT, and this involves a switch in the LSD1 histone methylation pattern: genome-scale mapping showed that DNA methylation patterns were unchanged and that chromatin changes were mainly specific to large organized heterochromatin K9 modifications60. Using epithelial prostate cells it was further demonstrated that global gene expression during EMT is regulated by the net intensity of active histone methylation minus repressive histone methylation and DNA methylation marks61. Intriguingly, there is no thorough understanding of the hierarchy, if one exists, of the different EMT-TFs, particularly because they are often studied individually or in limited groups, but rarely altogether. One straightforward way to do this is through the comparison of gene expression profiles in cells overexpressing the different NATURE REVIEWS | CANCER VOLUME 13 | FEBRUARY 2013 | 99 © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS Primary tumour site Homeostasis Normal epithelium SC Tumour progression Adenoma Therapy Invasive carcinoma Carcinoma in situ Recurrence Therapeutic CSC BM Metastatic site Resistant cell Parallel progression EMT Intravasation MET Blood vessel Extravasation Figure 1 | Role of EMT during cancer progression. In tumour cells, epithelial to mesenchymal transition (EMT)-inducing Nature Reviews | Cancer transcription factors (EMT-TFs) may primarily redefine the epithelial status of the cell, potentially — but not necessarily — assigning stem cell (SC) characteristics to dedifferentiated tumour cells, or they may redefine resident genetically altered stem cells to be cancer stem cells (CSCs). The dissemination of tumour cells from the solid tumour and subsequent migration after breakdown of the basement membrane (BM) — the classical view of the role of EMT in cancer — can only be achieved when all component pathways of the network are activated and fully parallels the process that is seen in development: if the cancer cell has acquired the necessary genetic aberrations and receives the appropriate signals at the tumour–host interface, the cell is ready to move towards metastasis. At this point, the active contribution of the EMT-associated programme is probably to give survival signals and to maintain the mesenchymal status of the metastasizing cell. It is likely that EMT also has a role in parallel progression, in which tumour cells escape early and metastasis progresses in parallel to the primary tumour. EMT features may further promote resistance during tumour therapy, leading to recurrence and a poor prognosis. The degree of EMT during the different steps in cancer progression probably depends on the imbalance of several associated regulatory networks with activated oncogenic pathways. MET, mesenchymal to epithelial transition. EMT-TFs or in the same cells in which these EMT-TFs are knocked down33,62,63. Unfortunately, a correct interpretation is impeded by a large number of pitfalls, such as the availability of compatible cofactors. However, knockdown experiments in several cancer cell lines with the co-expression of multiple EMT-TFs have shown that ablation of only one EMT‑TF can be sufficient to partially or totally block EMT and metastasis in experimental models28,64–66. In this case, the loss of expression of an EMT‑TF probably also induces the loss of a strong anti-apoptotic signal67 or the loss of associated stem-cell characteristics68,69. Nevertheless, the different EMT-TFs can drive common, as well as non-redundant, pathways: for example, loss of the newly characterized EMT‑TF PRRX1 enhances rather than suppresses stem-cell characteristics40, and thus its loss not only promotes MET but also allows outgrowth at the site of metastasis. Non-coding RNAs regulate EMT Small non-coding RNAs or microRNAs (miRNAs) are now commonly known as potent modifiers of gene expression that enable cells to rapidly respond to a new fate or environment. The number of miRNAs that has been directly or indirectly associated with EMT is becoming as extensive as the list of EMT-associated transcription factors70. Nevertheless, there is sufficient experi mental evidence to define two strong recurrent anchors in different cellular systems (TABLE 2). One is no doubt centred on the miR‑200 family 71,72, which has five members arranged as two clusters; mir‑200a, mir‑200b and mir‑429 on human chromosome 1, and mir‑200c and mir‑141 on human chromosome 12, with each cluster expressed as a polycistronic transcript 73. Expression of the miR‑200 family is strongly associated with epithelial differentiation, and a reciprocal feedback loop between the miR‑200 family and the ZEB family of transcription factors tightly controls both EMT and MET71,72. Importantly, this miRNA-mediated differentiation is not only due to ZEB targeting but also to regulation of the expression of accessory epigenetic machinery such as BMI1 and SUZ12 (REFS 69,74). SNAI1‑dependent EMT is directly controlled by miR‑34 family members75,76. Both miR‑200 and miR‑34 have E‑boxes in their promoters, which are directly regulated by SNAIL and ZEB transcription factors76,77, but the specificity, redundancy and/or cooperation between the two loops are poorly characterized. However, these loops seem to share similarity in both upstream inducers and operation modes, further creating the contours of a hierarchical framework that defines cellular plasticity. Moreover, additional miRNAs might provide additional support for the epithelial phenotype-like miR‑101, which maintains E‑cadherin expression by repressing EZH2 (REF. 78). 100 | FEBRUARY 2013 | VOLUME 13 www.nature.com/reviews/cancer © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS EMT MET Epigenetic machinery: • EZH2 • BMI1 Post-translational control Transcriptional control Differential splicing Non-coding RNA regulation • ELF3 • ELF5 • GRHL2 • SNAI1 • ZEB • TWIST1 • ESRP1 • ESRP2 RBFOX2 • miR-200 • miR-34 • miR-101 Epithelial features • E-cadherin • Polarity Post-translational control Transcriptional control Differential splicing Non-coding RNA regulation miR-9 • Vimentin • N-cadherin • Fibronectin Mesenchymal features Figure 2 | EMT is controlled by four major interconnected regulatory networks. Proteins involved in transcriptional Reviews Cancer control contribute to the best characterized regulatory network during epithelial to mesenchymal Nature transition (EMT).|Several EMT-inducing transcription factors, such as SNAI1, ZEB and TWIST1 have a central role in this network. Their function is further enhanced by the close cooperation with the epigenetic machinery that enables firm repression of epithelial features. Epithelial-specific transcription factors are far less studied in the context of EMT; they define the differentiation of the epithelial cell. Nevertheless, the epithelial status is dominated by the presence of several non-coding RNAs defining another strong network controlling EMT. A tight negative feedback loop is formed by the microRNAs (miRNAs) miR‑200 and miR‑34 with several EMT transcription factors (EMT-TFs). The epithelial phenotype is further supported by miR‑101, which promotes E‑cadherin expression. Non-coding RNAs at the mesenchymal side seem merely to support the mesenchymal phenotype. Differential splicing on both the epithelial and the mesenchymal sides is characterized by specific splicing factors, which determine specific epithelial or mesenchymal splice variants. Proteins involved in the different regulatory networks are further regulated on both translational and post-translational levels, either providing more support for or destabilizing their function. Currently, evidence for this regulatory level is almost exclusively available for EMT-TFs (solid arrows) and is less clear for transcription factors that maintain the epithelial phenotype (dashed arrows). Given the strong established interconnections between these regulatory layers, it is to be expected that future research will further reinforce this picture with additional regulatory circuits being found to exist between the molecules shown in the figure. EMT is initiated when the balance in these regulatory networks is disturbed (for example, by extracelluar signals or protein misregulation); the degree of EMT is further defined by how far the balance is tipped in one or the other direction. ELF3, E74‑like factor 3; ESRP1, epithelial splicing regulatory protein 1; GRHL2, Grainyhead-like 2; MET, mesenchymal to epithelial transition; RBFOX2, RNA binding protein FOX1 homologue 2. EMT-associated stem cell phenotypes Cells undergoing EMT acquire motility and stemness phenotypes. Both traits are believed to form the basis for dissemination and metastasis of differentiated carcinomas, as the plasticity allows permanent adaptations to the demanding conditions of a changing environment. Interestingly, the miR‑200 family has been shown to be downregulated in normal human and mouse mammary stem and progenitor cells79, a property shared with miR‑205 (REF. 80), another negative regulator of ZEB1 and ZEB2 (REF. 71). Downregulation of miR‑200 members has also been observed in malignant cancer stem cells in the colon, breast and prostate69,79,81. Both miR‑200s and miR‑34 are under the positive control of p53 (REFS 76,82–84). Although the underlying mechanisms for a precise role of p53 in stem cells are far from understood, it seems to be able to restrain stem-cell self-renewal and to impose an asymmetric mode of division, at least in breast tissue (reviewed in REF. 85). p53 deficiency facilitates the reprogramming of differentiated cells into iPS cells that closely resemble embryonic stem (ES) cells. Loss of p53 function has now been associated with the regulation of EMTassociated stem cell phenotypes and the expression status of stem-cell markers such as BMI1, CD44 and CD133 (REFS 76,82–84). Mutant p53 will not only prevent wild-type p53 from binding on the miR‑200 promoter 82 but will also directly repress miR‑130b, an inhibitor of ZEB1 (REF. 86). In a number of experimental models, mutant p53, rather then loss of p53, was found to augment the pro-migratory, pro-invasive and pro-metastatic properties of TGFβ in vitro and in vivo87. As such, mutant p53 may be involved in reinforcing the strong established network around TGFβ, ZEB transcription factors and miR‑200 (REFS 71,77), further facilitating our understanding of why recapitulation of EMT features in a cancer context might endow tumour cells with selected stem-cell characteristics68, enabling tumours to be formed more efficiently. The epigenetic regulation of the different epitheliumrelated miRs has recently received a lot of attention. There is an inverse correlation between mir‑200c and NATURE REVIEWS | CANCER VOLUME 13 | FEBRUARY 2013 | 101 © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS Table 1 | Molecular factors involved in EMT and cancer progression: transcriptional regulation Molecules Function and involvement in EMT Relevance to cancer Refs Transcription factors: direct binding to the CDH1 promoter SNAI1 Zinc-finger protein, transcriptional repressor and cellular reprogramming Upregulated in breast carcinoma, colon cancer, endometrial cancer, head and neck squamous cell carcinoma, hepatocarcinoma, gastric cancer, oesophageal adenocarcinoma, ovarian carcinoma, pancreatic carcinoma and synovial carcinoma 146,147 SNAI2 Zinc-finger protein, transcriptional repressor and cellular reprogramming Upregulated in breast carcinoma, colorectal carcinoma, oesophageal squamous cell carcinoma, gastric cancer, head and neck squamous cell carcinoma, hepatocarcinoma, lung adenocarcinoma, malignant mesothelioma, ovarian carcinoma and pancreatic cancer 147,148 ZEB1 Zinc-finger E‑box binding homeobox Upregulated in bladder cancer, breast cancer, colorectal cancer, endometrial protein, transcriptional repressor and adenocarcinoma, oesophageal squamous cell carcinoma, gastric cancer, head cellular reprogramming and neck squamous cell carcinoma, hepatocarcinoma, leiomyosarcoma, lung carcinoma, pancreatic cancer and prostate cancer 147 ZEB2 Zinc-finger E‑box binding homeobox protein, transcriptional repressor and cellular reprogramming Upregulated in bladder cancer, breast cancer, colorectal cancer, gastric cancer, head and neck squamous cell carcinoma, hepatocarcinoma, lung cancer, ovarian cancer and pancreatic cancer 147 E47 Class I basic helix–loop–helix (bHLH) factor Upregulated in gastric cancer and prostate cancer KLF8 Zinc-finger protein, transcriptional repressor and activator Upregulated in brain cancer, breast cancer, gastric cancer, liver cancer, ovarian cancer and renal cancer Brachyury Transcriptional activator Upregulated in colorectal cancer and lung cancer 149,150 151 152,153 Other transcription factors associated with EMT TWIST1 bHLH factor Upregulated in bladder cancer, breast cancer, colorectal cancer, oesophageal squamous cell carcinoma, gastric cancer, head and neck squamous cell carcinoma, hepatocarcinoma, ovarian carcinoma, pancreatic cancer and prostate cancer FOXC2 Transcriptional activator Upregulated in breast cancer, colorectal cancer, oesophageal squamous cell carcinoma and lung cancer Goosecoid Homeobox protein Upregulated in breast cancer E2‑2 Class I bHLH factor Upregulated in lung squamous cell carcinoma SIX1 Homeobox protein Upregulated in breast cancer, cervical cancer and ovarian cancer PRRX1 Homeobox protein Upregulated in breast cancer and lung cancer; correlated with lack of metastasis 147 36, 154–156 37 157 158–160 40 Accessory associated complexes and epigenetic machinery HDAC1, HDAC2 and HDAC3 Histone deacetylases Upregulated in breast cancer, colorectal cancer, gastric cancer, hepatocarcinoma, lung cancer, pancreatic cancer, prostate cancer and renal cell cancer 161 SIRT1 NAD-dependent histone deacetylase Upregulated in colorectal cancer and skin cancer 162 EZH2 PRC2 component Upregulated in B cell non-Hodgkin’s lymphoma, bladder cancer, breast cancer, colon cancer, glioblastoma, Hodgkin’s lymphoma, oral cancer, liver cancer, lung cancer, lymphoma, mantle cell lymphoma, melanoma, prostate cancer and testicular cancer 163 SUZ12 PRC2 component Upregulated in breast cancer, colon cancer, germinal cell-derived tumour, liver cancer, lung cancer, mantle cell lymphoma, melanoma, and parathyroid and pituitary adenoma 163 LSD1 H3K4 and H3K9 demethylase and epigenetic regulator Upregulated in breast cancer, bladder cancer, colorectal cancer, lung cancer, prostate cancer and sarcoma SUV39H1 Histone methyltransferase Upregulated in colorectal cancer 170 G9a Histone methyltransferase Upregulated in lung cancer 171 BMI1 PRC1 component Upregulated in acute myeloid leukaemia, breast cancer, gastrointestinal tumour, lymphoma, medulloblastoma, neuroblastoma, lung cancer, and parathyroid and pituitary adenoma 163 LOXL2 A lysyl oxidase Upregulated in colorectal cancer, breast cancer, laryngeal cancer, lung cancer, gastric cancer, oesophageal squamous cell cancer, ovarian cancer, head and neck carcinoma, and melanoma; and upregulated in stroma associated with colorectal cancer, pancreatic cancer, laryngeal cancer, endometrial cancer, testicular seminoma, hepatocarcinoma and renal clear cell cancer 164–169 172–174 EMT, epithelial to mesenchymal transition; EZH2, enhancer of zeste 2; FOXC2, Forkhead box C2; HDAC, histone deacetylase; KLF8, Krüppel-like factor 8; LOXL2, lysyl oxidase-like 2; LSD1, lysine-specific demethylase 1; PRC2, Polycomb repressor complex 2; PRRX1, paired-related homeobox gene 1; SIRT1, sirtuin 1; SUV39H1, suppressor of variegation 3–9 homologue 1; SUZ12, suppressor of zeste 12. 102 | FEBRUARY 2013 | VOLUME 13 www.nature.com/reviews/cancer © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS mir‑141 promoter methylation status and expression in breast and prostate cancer cell lines, which is accompanied by permissive and repressive histone modifications at this promoter corresponding to an epithelial and mesenchymal status, respectively 88. Promoter hypermethy lation of mir‑205 has also been detected in prostate cancer 89. Epigenetic silencing of the mir‑200 family and mir‑205 has thus been associated with early transformation of epithelial cells90. In colorectal cancer, miR‑200 expression is lower in the primary tumour, particularly at the invasive front 91,92, whereas higher expression is again seen in corresponding metastases, associated with hypomethylation of the promoter region92. Consistent with this, it was shown that overexpression of miR‑200 in weakly metastatic breast cancer cells suppresses SEC23A‑mediated secretion, and that this promotes further metastatic colonization of these cells in distant organs in mice, suggesting that the role of the miR‑200 family in metastasis not only relies on promoting MET but also on altering other cellular properties such as the secretome, which can influence the microenvironment to permit metastasis93. The miR‑200 and miR‑34 family members are strongholds of the epithelial and differentiated phenotype, and thus are considered to be tumour suppressive miRNAs, whereas there is also evidence for miRNAs promoting the transition to a mesenchymal phenotype (TABLE 2). However, the expression pattern for many — if not all — miRNAs is often cell-context specific. miR‑9 targets CDH1 directly, but it does not necessarily induce EMT94. Similarly, increased miR‑92a expression will reduce CDH1 expression, resulting in the promotion of carcinoma cell motility and invasiveness95. It is likely that these miRNAs that are associated with the mesenchymal phenotype are downstream effectors fine-tuning the general cellular reprogramming. This was also shown for miR‑99a and miR‑99b: although their inhibition was not sufficient to block TGFβ-induced EMT, their expression could contribute to migration and lower E-cadherin and tight junction protein ZO1 expression96. Although overwhelming evidence supports the role of different miRNAs during EMT, there is much less known about other classes of non-coding RNAs. We are only beginning to understand their complexity and nature, leaving an emerging field in RNA biology, probably with important implications for EMT. A notable example was the description of a natural antisense transcript that is upregulated after SNAI1‑induced EMT and prevents the processing of a large intron that is necessary for the expression of ZEB2 (REF. 97), clearly illustrating the possibility that EMT transcripts can be present without the need to be translated into protein. This observation may also help us to understand the recent finding that the ZEB2 transcript functions as a competitive endogeneous RNA (ceRNA) for PTEN miRNAs. The aberrant regulation of PTEN following ZEB2 loss can contribute to melanoma development 98. This described role of ZEB2 as a tumour suppressor may be counterintuitive at first glance, but it fits well with the idea that deregulation of a transcription factor with a potential important function in a cell type other than the epithelium may be involved in tumour progression. EMT-associated differential splicing Besides the well-established transcriptional reprogramming during EMT, post-transcriptional mechanisms such as alternative pre-mRNA splicing also provide an additional layer of gene regulation that further enhances EMT. The close connection between EMT and alternative splicing events was first shown for fibroblast growth factor receptor 2 (FGFR2), the protooncogene RON (also known as macrophage stimulating Table 2 | Molecular factors involved in EMT and cancer progression: non-coding RNAs Molecules Function and involvement in EMT Relevance to cancer Refs miR‑200 family Tumour suppressor function Downregulated in bladder cancer, gastric cancer, spindle cell carcinoma of the head and neck, ovarian cancer and endometrial carcinosarcoma 142, 175–178 Upregulated in endometrial endometrioid carcinoma, pancreatic cancer; higher expression in distant metastasis versus primary breast cancer; and increased levels in blood of patients with gastric cancer 93, 179–184 Downregulated in bladder cancer, melanoma, spindle cell carcinoma of the head and neck, head and neck squamous cell carcinoma and prostate cancer 177,178, 185–189 miR‑205 Tumour suppressor function miR‑34 Tumour suppressor function Downregulated in breast cancer, colorectal cancer, lung cancer, ovarian cancer, pancreatic cancer, sarcoma, renal cell cancer and urothelial cancer 191–193 miR‑101 Tumour suppressor function Downregulated in gastric cancer, hepatocellular carcinoma and lung cancer 194–197 miR‑9 Tumour promotor function Upregulated in brain cancer, breast cancer and gastric cancer; downregulated in breast cancer and ovarian cancer; higher expression in distant metastasis versus primary breast cancer; lower expression in metastatic versus primary brain tumours 179,198 Upregulated in lung cancer and endometrial cancer 190 EMT, epithelial to mesenchymal transition; miR, microRNA. NATURE REVIEWS | CANCER VOLUME 13 | FEBRUARY 2013 | 103 © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS Exon junction arrays Microarrays constructed to measure the expression of alternative splice forms of a gene. The probes used are specific for expected or potential splice sites of predicted gene exons. Cap-independent translation The internal ribosome entry site (IRES) in the mRNA 5ʹ untranslated region is used for translation initiation. Cap-independent mRNAs can bypass translation inhibition when subjected to stresses such as hypoxia, and some mRNAs can use both the cap and IRES. 1 receptor (MST1R)) and CTNND1 (which encodes p120 catenin)34,99,100, suggesting that the balance between specific splicing isoforms is relevant, whether or not EMT will occur. For example, the exon 11 skipping of RON is enhanced in post-EMT cells and results in a constitutively active tyrosine kinase receptor that confers invasive properties. The production of this alternative RON transcript is regulated by the proto-oncogene and splicing factor serine/arginine-rich splicing factor 1 (SRSF1) the expression level of which is attenuated by SAM68 in epithelial cells. The level and/or phosphorylation status of the splicing regulator SAM68 exerts its effect in epithelial cells by non-productive splicing in the 3ʹ untranslated region (UTR) of SRSF1, resulting in alternative splicing-activated nonsense-mediated mRNA decay 99,101. Two paralogous RNA binding proteins, epithelial splicing regulatory protein 1 (ESRP1) and ESRP2, were identified as essential promoters of the epithelial pattern of FGFR2 splicing in a highthroughput cell-based cDNA expression screen102. These factors also control the specific splicing of the epithelial isoforms of CTNND1, CD44 and the orthologue of mammalian enabled MENA (also known as ENAH). The potential functional contribution towards the EMT phenotype of these spliced genes is nicely exemplified by CTNND1: the epithelium-specific splicing of CTNND1 results in a shorter isoform of the protein that promotes cell–cell adhesion by stabilizing E‑cadherin at the plasma membrane, whereas the longer mesenchymal isoform induced during EMT binds RHOA and attenuates its activity, resulting in enhanced cellular invasion103. Although this demonstrates the potential potent consequences of reduced ESRP expression, it should be clear at this stage that EMT-related differential splicing events occur as part of a general cellular reprogramming, and that the specific functional contributions of different splice isoforms are not yet clear in most cases. To determine a genome-wide ESRP-regulated alternative splicing network, the functional consequence of either ESRP knockdown in cells with an epithelial differentiation pattern or ectopic ESRP expression in MDA‑MB‑231 cells (post-EMT) was measured using exon junction arrays. This resulted in the discovery of more than 200 validated ESRP-enhanced or ESRP-silenced exon splicing changes104. On the basis of these well-defined splicing events, the search for conserved sequences in the exons and flanking introns resulted in the identification of enriched motifs with UGG as a core motif. ESRP binding in the exon and/or in the upstream intron drives exon skipping, whereas ESRP binding downstream of the exon promotes exon inclusion. Thus, the position of ESRP UGG-rich binding motifs determines an epithelial splicing regulatory ‘RNA map’. Disruption of the epithelial splicing programme through ESRP knockdown in human mammary epithelial cells enables these cells to change morphology and to become more motile. Mesenchymal markers such as vimentin, fibronectin and N‑cadherin are induced, whereas no changes of epithelial markers such as E‑cadherin were detected. Importantly, ESRP genes are directly downregulated by the EMT-inducing transcription factors SNAI1, ZEB1 and ZEB2 (REFS 105,106). In addition to ESRP1 and ESRP2, factors belonging to the RNA binding protein FOX1 homologue (RBFOX), CUGBP Elav-like family (CELF), muscleblind-like protein (MBNL) and heterogeneous nuclear ribonucleoprotein (HNRNP) families of tissue-specific factors have been identified as being involved in the regulation of EMT-specific splicing 107. This was done using transcriptome deep-sequencing of a TWIST1‑induced EMT model in mammary epithelial cells and subsequent detailed motif analysis of regions adjacent to the splice sites in EMT-regulated alternative transcripts. The importance of this more complex view of EMT-associated alternative splicing was shown in dedifferentiated mammary epithelial cells, which on knockdown of RBFOX2 shifted to a more epitheliallike morphology that was marked by reduced vimentin expression and stress-fibre presence, and by the reapp earance of epithelial junctional markers such as ZO1 and α-catenin at cell–cell contacts107. These experiments are consistent with an earlier report that the claudin-low subtype of breast cancer (which is enriched for markers of EMT) has a specific alternative splicing pattern that is associated with evolutionarily conserved binding motifs for the RBFOX2 splicing factor 108. Together, these findings suggest a model in which a series of splicing factors collaborate to mediate splicing through position-dependent RNA binding as part of a functional coherent splicing regulation network (FIG. 3). Furthermore, the findings raise the intriguing possibility that abnormal changes in epithelial-specific splicing can steer cancer cells towards malignant progression through a partial EMT, without the need for canonical transcriptional reprogramming. The relevance of EMT-associated alternative splicing events is supported by the low expression of ESRP1 and ESRP2 in EMT-like cancer cell lines and by the finding that luminal B and basal breast cancer cell lines can be distinguished using splicing patterns alone, supporting the idea that EMT-associated alternative splicing isoforms could be used to potentially classify human breast cancer104,107. Translational and post-translational regulation The post-transcriptional miRNA regulatory loops that control the translation of SNAIL and ZEB family members are well established, but more recently it has been shown that general translational regulatory mechanisms also have a substantial impact on EMT. Y‑Box binding protein 1 (YB1; also known as nucleasesensitive element-binding protein 1) is a general mRNA binding protein that, at higher concentrations in the cell, represses translation and facilitates the storage of 5ʹ 7‑methylguanosine cap-containing mRNAs. Increased expression of YB1 has been associated with malignant cancer progression (TABLE 3). In RAS-transformed breast epithelial cells, YB1 stimulates cap-independent translation of mRNAs through internal ribosome entry site (IRES)driven translation initiation, including those encoding the EMT-associated transcription factors SNAI1, ZEB2, lymphoid enhancer binding factor 1 (LEF1) and TWIST1. As such, translational regulation by YB1 will not only induce EMT in those cells but it will also potentially enhance the metastatic capacity 109. 104 | FEBRUARY 2013 | VOLUME 13 www.nature.com/reviews/cancer © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS Exon inclusion + ESRP EBSE Epithelium-specific splicing Non-epithelium-specific splicing Exon skipping ESRP – EBSI – ESRP EBSI Epithelium-specific splicing Non-epithelium-specific splicing Exon inclusion + RBFOX2 RBSE Mesenchyme-specific splicing Non-mesenchyme-specific splicing Figure 3 | EMT is controlled by differential splicing events. Trans- and cis-acting Nature Reviews elements controlling epithelium- and mesenchyme-specific splicing are shown.| Cancer Epithelial splicing regulatory protein 1 (ESRP1) and ESRP2 regulate epithelium-specific splicing by recognition and binding of UGG- or GGU-rich RNA sequences that are known as ESRP binding splicing enhancer (EBSE) and ESRP binding splicing inhibitor (EBSI). Exon inclusion or skipping is determined by the location of the EBSE and EBSI elements relative to the alternatively spliced exons. ESRP binding to EBSI favours exon skipping when its binding sites are located at the 5ʹ end of, and/or within, the regulated exon motifs, whereas ESRP proteins binding at the 3ʹ end of regulated exons enhance exon inclusion. The splicing factor RNA binding protein FOX1 homologue 2 (RBFOX2) was shown to control mesenchyme-specific splicing. RBFOX2 binds to the UGCAUG RNA sequence known as RBFOX2 binding splicing enhancer (RBSE) that is found downstream of alternatively spliced exons and that favours the inclusion of the alternative exon in the mature mRNA. Examples for epithelium- and mesenchymespecific splicing are shown for generic genes: exons are shown as boxes, and introns are shown as horizontal lines; alternatively spliced regions are shown in blue. EMT, epithelial to mesenchymal transition. Ribosomal A site During elongation cycles of translation each aminoacyl-tRNA enters the ribosome at the A site where the match is tested for the tRNA anti-codon with the codon of the mRNA, and where the amino end of its amino acid is transferred to the carboxylic end of the nascent chain. In addition to transcriptional induction of EMT‑TF and activation of the miR‑200 family, TGFβ also controls EMT through the regulation of a crucial translational checkpoint at the elongation stage of EMT effector transcripts. Blocking of translational elongation can be accomplished with HNRNPE1 (also known as PCBP1), which inhibits the release of elongation factor 1α1 (EEF1A1) from the ribosomal A site. The interaction between HNRNPE1 and EEF1A1 is stabilized by binding to the stem–loop structural 33‑nucleotide-long BAT (TGFβ-activated translation) element that is present in the 3ʹ UTR of regulated mRNAs. On TGFβ signalling, the BAT–HNRNPE1–EEF1A1 complex is disrupted by AKT2‑mediated HNRNPE1 phosphorylation. It thereby reactivates translational elongation of target EMT transcripts, such as interleukin-like EMT inducer (ILEI) and disabled homologue 2 (DAB2), which are both involved in malignant cancer progression110. Most other experimental information on posttranslational regulation of EMT is available for the EMT‑TF SNAI1, which is known to have a fast turnover time in the cytoplasm. The importance of avoiding abnormal SNAI1 protein expression is reflected by a multitude of fine-tuned regulatory mechanisms such as post-translational phosphorylation, lysine oxidation and ubiquitylation1. In general, these regulatory mechanisms can be separated into glycogen synthase kinase 3β (GSK3β)-dependent and GSK3β‑independent mechanisms of SNAI1 regulation. GSK3β is a serine/threonine kinase that functions as a central regulator in canonical WNT signalling and has been found to be essential for the maintenance of epithelial differentiation111. In the context of SNAI1 clearance, casein kinase 1 (CK1) functions as a priming kinase; the phosphorylation of SNAI1 by CK1 is required for subsequent GSK3β phosphorylation of SNAI1 at serine/threonine residues that are then recognized and ubiquitylated by the E3 ubiquitin ligase βTRCP1 for degradation112–114. This mechanism is mostly counteracted by several receptor tyrosine kinase signalling pathways that stimulate SNAI1 protein stability by inactivating GSK3β. Likewise, inflammatory cytokine tumour necrosis factor‑α (TNFα)-mediated SNAIL stabilization is realized through nuclear factor-κB (NF‑κB), which drives β-casein (CSN2)-controlled disruption of the binding of GSK3β with SNAI1 and βTRCP1 (REF. 115). The WNT–GSK3β–βTRCP1 axis was also found to regulate SNAI2 activity that controls cancer cell EMT programmes and to coordinately reduce BRCA1 expression116. GSK3β‑independent ubiquitin ligases, such as MDM2 and FBXL14, have also been reported to target SNAI1 and SNAI2 for degradation 117–119 (TABLE 3). Interestingly, it was recently reported that the F‑box protein partner of paired (PPA) targets SNAI1, SNAI2, TWIST1 and ZEB2 for degradation, suggesting that there is coordinated control of these structurally unrelated core nuclear factors implicated in EMT120. Conversely, SNAI1 protein is stabilized by specific phosphorylation by the kinases p21-activated kinase 1 (PAK1) and ataxia telangiectasia mutated (ATM)121,122. Notably, these EMT-inducing kinases are activated by DNA damage. Alternatively, direct interaction between LOXL2 and SNAI1 stabilizes this major EMT-TF57. In addition, hyperglycaemic conditions driving O‑β-d-Nacetylglucosamine (O-GlcNAc) modifications of SNAI1 enhance its stability 123. By contrast, increased expression of proteases, which are detected in nearly every cancer type, can promote EMT induction without directly affecting EMT-TFs but instead by modulation of signal transduction leading to stabilization of EMT. For example, simple treatment of mammary epithelial cells with exogenous matrix metalloproteinase 3 (MMP3) can result in a RAC1B‑mediated increase of reactive oxygen species, which corresponds with increased levels of SNAI1 and mesenchymal markers124. In a similar manner, MMP13 and transmembrane protease serine 4 (TMPRSS4) affect EMT by indirect enhancement of EMT-TF activity 125,126. Although MMPs have a direct role in extracellular matrix (ECM) degradation, EMT can also promote this process, as it was shown that TWIST1 is capable of promoting the formation of invadopodia, which are specialized membrane protrusions for ECM degradation127. Likewise, invadopodium-mediated proteolysis takes place at the vascular basement membranes128. Interestingly, tumourassociated macrophages physically help tumour cells NATURE REVIEWS | CANCER VOLUME 13 | FEBRUARY 2013 | 105 © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS during this process by creating a microenvironment that is permissive for invasion through epidermal growth factor (EGF) and macrophage colony-stimulating factor 1 (CSF1) feedback signals129. As such, the study of the role of macrophages and, more generally, of accessory cells in driving tumour-cell motility may uncover important underlying pathways in which crosstalk with EMT-regulatory proteins cannot be excluded. Several lines of evidence support the idea that active regulation of SNAI1 subcellular localization can control nuclear activity. Furthermore, the subcellular localization of SNAI1 has a direct influence on its turnover rate, as nuclear SNAI1 degrades more slowly than cytosolic SNAI1 (REF. 114). The kinases PAK1 and LATS2, which are found to be overexpressed in different epithelial cancers, phosphorylate SNAI1, favouring nuclear retention, thus enhancing both stability and transcriptional activity of SNAI1 (REFS 122,130) (TABLE 3). In addition, the zinc transporter LIV1 (also known as ZIP6), a signal transducer and activator of transcription 3 (STAT3) target, controls the nuclear import of the SNAI1 protein131. In this context it is worthwhile to mention that TAZ, a transcriptional coactivator that is regulated by the Hippo tumour suppressor pathway, is phosphorylated by LATS2, attenuating its ability to induce EMT132. This inhibition of TAZ in epithelial cells seems to depend on its association with the cell-polarity determinant Scribble. Loss of Scribble activates TAZ, and this endows epithelial cells with selfrenewal capabilities and links EMT-like processes with the induction of cancer stem cell properties133. Finally, a plausible explanation for the apparent opposing effects of LATS2 on EMT is dependent on the cellular localization of LATS2: SNAI1 is a nuclear target and TAZ is a cytoplasmic target134. Export out of the nucleus is initiated by protein kinase D1 (PRKD1), which phosphorylates SNAI1, enabling it to bind with the epithelial specific 14‑3‑3σ complex that shuttles it out of the nucleus. Notably, PRKD1 expression is attenuated in different carcinoma types and its re‑expression in prostate cancer results Table 3 | Molecular factors involved in EMT and cancer progression: alternative splicing and post-translational regulation Molecules Function and involvement in EMT Relevance to cancer Refs Alternative splicing ESRP1 and ESPR2 mRNA splicing factor regulating the formation of epithelial cell-specific isoforms Reduced expression in claudin-low and metaplastic breast cancer 104 RBFOX2 RNA binding protein that regulates alternative splicing events Not known 107 Post-translational regulation YB1 Translation stimulation or repression dependent on YB1/mRNA ratio Overexpression in breast cancer cells induces EMT 109 HNRNPE1 Blocking translational elongation of EMT trancripts by binding to the 3ʹ UTR structural element BAT. Phosphorylated by AKT2 on TGFβ signalling, resulting in reactivated translation Repression in breast cancer cells induces EMT 199 CK1 Serine/threonine protein kinase that primes SNAI1 for GSK3β‑mediated degradation Repression in cells induces EMT 112 CSN2 TNFα-mediated block of SNAI1 ubiquitylation and degradation by inhibiting SNAI1 binding with βTRCP and GSK3β Activation in breast cancer cells on TNFα exposure results in strengthening of the invasion and migratory phenotype 115 MDM2 p53 E3 ubiquitin protein ligase that degrades SNAI2 Reduced expression in non-small-cell lung cancer results in high SNAI2 expression correlated with poor patient survival 119 FBXL14 Substrate-recognition component of some SCF (SKP1– CUL1–F‑box protein)-type E3 ubiquitin-protein ligase complexes interacts with SNAI1 and promotes its ubiquitylation and proteasome degradation Decreased expression in hypoxic colorectal tumours 117 PAK1 Serine/threonine-protein kinase that phosphorylates SNAI1, resulting in nuclear localization Enhanced expression in gastric cancer, non-small-cell lung cancer, head and neck cancer, and breast cancer LATS2 Serine/threonine protein kinase that retains SNAI1 nuclear localization Enhanced expression in breast cancer LIV1 STAT3‑induced zinc transporter controlling nuclear import of SNAI1 Enhanced expression in oestrogen receptor-positive breast cancer and linked with its metastasis PRKD1 Suppresses EMT through control of SNAI1 nuclear export Downregulated in prostate cancer, breast cancer and gastric cancer 135 CalDAG-GEFIII Binds ZEB1 in cytoplasm, which activates RAS and attenuates angiogenesis Not known 137 122, 200,201 130 131,202 CalDAG-FEFII, calcium and diacylglycerol-regulated guanine nucleotide exchange factor 1; CK1, casein kinase 1; CSN2, β-casein; EMT, epithelial to mesenchymal transition; ESRP1, epithelial splicing regulatory protein 1; FBXL14, F-box and leucine-rich repeat protein 14; GSK3β, glycogen synthase kinase 3β; HNRNPE1, heterogeneous nuclear ribonucleoprotein E1; LATS2, large tumour suppressor 2; LIV1, solute carrier family 39, member 6; PAK1, p21 protein-activated kinase 2; PRKD1, protein kinase D1; RBFOX2, RNA binding protein FOX1 homologue 2; STAT3, signal transducer and activator of transctiption 3; TGFβ, transforming growth factor-β; TNFα, tumour necrosis factor-α; UTR, untranslated region. 106 | FEBRUARY 2013 | VOLUME 13 www.nature.com/reviews/cancer © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS in MET with clear repression of mesenchymal markers and diminished metastatic capability 135 (TABLE 3). A tight control of subcellular localization might also be involved in the regulation of ZEB transcription factors, as phylogenetic domain analysis revealed the probable presence of well-conserved nuclear import and export signals, suggesting active regulation136. Indeed, in addition to its nuclear localization ZEB1 has been found in the cytoplasm, where it interacts with RAS guanylreleasing protein 3 (calcium and DAG-regulated) (RASGRP3) to modulate adhesion-induced activation of RRAS in endothelial cells137. ZEB transcription factors can also be sumoylated, disrupting the recruitment of the co‑repressor C-terminal binding protein (CTBP). ZEB2 sumoylation has been shown to result in attenuated transcriptional repression of CDH1 (REF. 138). 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Acta 1611, 16–30 (2003). Acknowledgements The authors apologize to those colleagues whose relevant studies were not cited here. The authors’ research is funded by grants from the VIB; the Fonds Wetenschappelijk Onderzoek, the geconcerteerde onderzoeksacties of Ghent University, Belgium; the Stichting tegen Kanker; the Association for International Cancer Research, UK; and the EU‑FP7 framework program TuMIC 2008–201662. Competing interests statement The authors declare no competing financial interests. FURTHER INFORMATION Geert Berx’s homepage: http://www.dmbr.ugent.be/index.php?id=geertberxhome ALL LINKS ARE ACTIVE IN THE ONLINE PDF www.nature.com/reviews/cancer © 2013 Macmillan Publishers Limited. All rights reserved