Oncogene (2014) 33, 1755–1763 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc REVIEW Multilayer control of the EMT master regulators H Zheng and Y Kang Metastasis is the leading cause of cancer-associated death in most tumor types. Metastatic dissemination of cancer cells from the primary tumor is believed to be initiated by the reactivation of an embryonic development program referred to as epithelial– mesenchymal transition (EMT), whereby epithelial cells lose apicobasal polarity and cell–cell contacts, and gain mesenchymal phenotypes with increased migratory and invasive capabilities. EMT has also been implicated in the regulation of cancer stem cell property, immune suppression and cancer regression. Several transcription factors have been identified as master regulators of EMT, including the Snail, Zeb and Twist families, and their expression is tightly regulated at different steps of transcription, translation and protein stability control by a variety of cell-intrinsic pathways as well as extracellular cues. Here, we review the recent literature on the signaling pathways and mechanisms that control the expression of these master transcription factors during EMT and cancer progression. Oncogene (2014) 33, 1755–1763; doi:10.1038/onc.2013.128; published online 22 April 2013 Keywords: cancer metastasis; EMT; Snail; Twist; Zeb; E3-ubiquitin ligase EMT AND CANCER METASTASIS embryogenesis, EMT has a key role in the formation of various Ninety percent of cancer-related deaths are not caused by tissues and organs such as the neural crest, heart, musculoskeletal 7–9 the growth of primary tumors but rather by the spread of and peripheral nervous systems. In adult organisms, only a cancer cells to distant secondary organs.1–3 Despite its devastating certain subset of cells retains the ability to undergo EMT in specific consequence, cancer metastasis is in fact a very inefficient process physiological or pathological conditions, such as wound healing. that requires tumor cells to overcome a series of challenging However, during cancer progression, tumor cells often gain the hurdles as they spread from the primary tumor to secondary ability to reactivate the EMT program and use it for their own 12 organs.4,5 During the expansion of the primary tumor, benefits. Several in vitro cell culture models and mouse models accumulating genetic and epigenetic changes, as well as tumor of cancer progression have shown that reactivation of EMT by microenvironment cues, cause a small portion of tumor cells to tumor cells confers selective advantages, including enhanced disseminate from the tumor mass, invade through the basement motility and invasiveness during tumor progression that is membrane, escape the host immune surveillance, enter systemic believed to be pre-requisite for tumor cell dissemination and 7–9,13 circulation, and spread to secondary organs. Disseminated tumor metastasis. cells then extravasate through the capillary endothelium into surrounding tissues, find a way to survive in the new environment and eventually grow to become clinically detectable DYNAMICS OF EMT AND MESENCHYMAL-TO-EPITHELIAL macrometastases. TRANSITION IN METASTASIS As an early step in cancer metastasis, tumor cells need to gain In tumor biology, EMT has been extensively studied using in vitro their ability to disseminate from solid tumor mass and invade into cell line models, but direct observation of EMT in vivo, particularly the surrounding stromal tissues either as a group of cells (cohesive in clinical specimens, has been difficult and there has been migration) or as single cells (mesenchymal invasion or amoeboid skepticism regarding its pathological relevance.14,15 Analysis of invasion).6 As epithelial tumor cells are usually tightly associated clinical patient samples have demonstrated features of EMT with their neighboring cells via E-cadherin-containing adherens among cancer cells in the invasive front of a number of different junctions, tumor cells must break these intercellular junctions tumor types,16–19 but success in identifying EMT-transitioning cells before they can move out as single cells and invade stromal in vivo has been hampered by the spatial and temporal tissues. Epithelial tumor cells may undergo a process termed as heterogeneity of EMT in human cancer and lack of reliable EMT epithelial-to-mesenchymal transition (EMT) to facilitate this type of markers that can distinguish tumor cells having undergone EMT cellular invasion (Figure 1), which can be stimulated either by from surrounding stromal cells.20 One explanation for this is that extracellular cytokines, such as TGF-b, EGF, FGF, or by intracellular cancer cells only undergo a transient EMT, reverting back to the cues, such as oncogenic Ras or NFkB signaling.7–11 During EMT, epithelial state by mesenchymal-to-epithelial transition (MET) epithelial cells lose their epithelial polarity and cell–cell contacts once they have penetrated stromal tissue, which facilitates and gain mesenchymal phenotypes with increased migratory and colonization in the target organs.21 Indeed, recent experimental invasive capabilities. A hallmark of EMT is the functional loss of evidence showed that although EMT can increases tumor invasion E-cadherin, while additional cellular changes, such as reduced and cancer stem cell properties,22 tumor cells undergoing EMT are expression of epithelial markers cytokeratins and ZO-1, and the often growth arrested since many EMT-inducing transcriptional overexpression of mesenchymal markers N-cadherin, Vimentin factors can directly inhibit proliferation.16,23–27 Thus, tumor cells and Fibronectin, are also often observed. During metazoan must undergo MET to revert to the epithelial state to allow Department of Molecular Biology, Princeton University, Princeton, NJ, USA. Correspondence: Professor Y Kang, Department of Molecular Biology, Princeton University, LTL255, Washington Road, Princeton, NJ 08544, USA. E-mail: [email protected] Received 9 February 2013; revised 27 February 2013; accepted 27 February 2013; published online 22 April 2013 Multilayer control of the EMT master regulators H Zheng and Y Kang 1756 metastatic growth in distant organs. The mechanisms underlying cancer—perhaps the seeds of metastasis have disseminated years the induction of MET are largely unknown but it is suspected that before the clinical detection of primary pancreatic cancer. withdrawal of EMT inducers, resulting from changes in the stromal In aggregate, recent advances in clinical and experimental studies cues provided by the surrounding environment, is a potential have firmly established the pathological relevance of EMTs in cause of MET. In support of the sequential EMT/MET model, cancer metastasis and underscore the importance of elucidating dynamic expression of E-cadherin has been documented in cancer molecular mechanism that controls such transitions. progression.28 Whereas those cells that undergo hypermethylation and repression of CDH1 in primary breast cancers are more invasive and metastatic, subsequent TRANSCRIPTION FACTORS IN EMT demethylation and reactivation of CDH1 during the process of As a crucial ‘gate-keeper’ of epithelial characteristics, E-cadherin is metastasis is observed. Consistent with the dynamic expression of mutated or downregulated in many cancer types and is associated CDH1, it has been reported that E-cadherin levels are elevated in with increased invasive behavior of tumor cells.8 For example, in lymph-node metastases relative to matched primary tumor breast cancer, at least 6% of breast cancer patients’ genomes samples, suggesting once again that EMT in primary tumors is 37 29–31 harbor E-cadherin null mutations, which permanently inactivate followed by MET at distant sites. Recently, multiple studies in the function of E-cadherin. Reversible suppression of E-cadherin is experimental mouse models demonstrated that tumor cells need mediated by epigenetic silencing, as well as a network of to downregulate EMT inducers and regain epithelial phenotype transcriptional repressors that target the CDH1 gene promoter.38 before they can form metastatic colonies in distant organs, These EMT transcriptional repressors include Snail (Snail1),39,40 indicating that a complete EMT–MET cascade is important for 41 13 42 43,44 45 46 23,32–35 Snail2 (Slug), Twist1, Zeb1, Zeb2, Goosecoid, FOXC2, tumor metastasis. FoxQ1,47 KLF8,48 Prrx132 and many others. In this review, we will EMT and tumor dissemination have long been believed to be focus on the Snail, Twist and Zeb families of transcription factors initiated at the advanced stage of tumor progression when and provide a focused discussion of the molecular network that changes of tumor microenvironment such as hypoxia and regulate their activities in controlling EMT and cancer metastasis. inflammation induce the invasive behavior of malignant cells. However, this concept has recently been challenged by an elegant animal model in which lineage tracking was used to identify The Snail zinc finger transcription repressor family epithelial cells that have undergone EMT and disseminate to Snail family members are the most intensively studied EMT distant organs.36 In this study, EMT and cell dissemination can transcriptional repressors. A wide ranges of signaling pathways be found at pre-neoplastic stage of pancreatic tissues, and have been found to induce Snail1 and Snail2 expression including pathological conditions such as pancreatitis dramatically TGF-b,49–51 Notch52,53 and Wnt pathways,54–56