Abstract The epithelial–mesenchymal transition (EMT) provides an outstanding example of cellular plasticity during embryonic development and cancer progression. During EMT in embryonic development, epithelial cells lose all vestiges of their epithelial origin and acquire a fully mesenchymal phenotype, known as complete EMT, which is typically characterized by a so-called cadherin switch. Conversely, during EMT in cancer progression, cancer cells that originate from epithelial cells exhibit both mesenchymal and epithelial characteristics, that is the hybrid E/M phenotype in a process known as partial EMT. Partial EMT in cancer cells is thought to enhance their invasive properties, generate circulating tumour cells and cancer stem cells, and promote resistance to anti-cancer drugs. These phenotypic changes are regulated by extracellular matrix components, exosomes and soluble factors, which regulate several transcription factors known as EMT transcription factors. In this review, I summarize our current understanding of the EMT program during cancer progression. cancer, differentiation, E-cadherin, EMT, invasion Epithelial–mesenchymal transition (EMT), a phenotypic conversion that facilitates embryonic development, was initially described by developmental biologists (1). EMT is a process by which epithelial cells lose their junctions and polarity, producing migratory mesenchymal cell types, such as, mesoderm and neural crest, during embryogenesis (2). The epiblast cells of the primitive ectoderm undergo EMT during the gastrulation stage to differentiate into primary mesenchymal cells. EMT converts some cells of each palatal shelf at the time of palatal fusion in the oral cavity into mesenchyme, leading to the formation of the secondary palate to produce the correct separation of oral and nasal cavities in mammals (3–5). Conversely, mesenchymal cells during embryonic development of the kidneys undergo mesenchymal–epithelial transition (MET) to differentiate into renal epithelium, ultimately resulting in nephrogenesis (6). In adults, the EMT program plays crucial roles in pathophysiological processes, including wound healing, tissue fibrosis and cancer progression (7, 8). After injury to the skin, neighbouring keratinocytes undergo an activation process characterized by molecular, morphological, cytoskeletal, and adhesive changes and start to migrate toward the wound centre (9). Some of these changes resemble those that occur during EMT. EMT is also associated with heart regeneration after injury and the ovarian surface epithelium. Chronic progressive fibrosis occurs in the kidney and liver, because of a variety of primary illnesses such as diabetes mellitus, hypertension and toxic injury, leading to impairment of their proper function. The epithelial cells, including endothelial cells, of these injured tissues in both acute and chronic disease undergo EMT and endothelial–mesenchymal transition (EndoMT), which have been confirmed by numerous independent studies, including animal models of chronic diseases. By utilizing a sophisticated mouse model with the indisputable identification of cells derived from the epithelium, approximately 30% of fibroblasts in fibrosis were determined to be derived from the epithelium via EMT (10). On the other hand, EMT has also been studied extensively in cancer, and is believed to play a crucial role in invasion, metastasis, chemo-resistance and processes that are involved in cancer cell aggressiveness (11). Partial EMT in Cancer Progression Cellular plasticity during cancer progression is the transition between epithelial and mesenchymal phenotypes, EMT and its reverse, MET. EMT involves dramatic cellular changes in which epithelial cells loosen their attachments to neighbouring cells, loses apico-basal polarity, become elongated and display increased motility, forming the first step of the invasion–metastasis cascade (12, 13). EMT is, in fact, accompanied by dissolution of adherens junction proteins and disruption of tight junctions, resulting in dissociation of epithelial cells. Conversely, EMT induces expression of mesenchymal marker proteins, and facilitates both attachment to the extracellular matrix (ECM) and acquisition of mesenchymal features such as spindle-shaped cell morphology and reorganization of actin stress fibres, thereby gaining the ability to migrate individually and invade through basement membranes and blood/lymphatic vessel walls. After intravasation, the cells undergoing EMT survive in the bloodstream as circulating tumour cells (CTCs), and finally extravasate into distant organs (14). When generating metastases, cancer cells are thought to undergo the MET to regain their epithelial characteristics and form secondary tumours. Besides enhancement of their motile properties, cancer cells undergoing EMT exhibit more aggressive phenotypes, including resistance to anti-cancer drugs and various stresses, inhibition of senescence and anoikis, and acquisition of immunosuppression and cancer stem cell (CSC)-like features (15, 16). Although cells undergo complete EMT during embryogenesis, which is represented by the cadherin switch, cancer cells express epithelial and mesenchymal markers (epithelial/mesenchymal (E/M) phenotype) concurrently, in what is termed partial EMT (17). In fact, CTCs that survive in blood exhibit E/M phenotypes, become resistant to anoikis, and exit the bloodstream more efficiently (18). In addition, effective metastasis is caused by cancer cells without complete loss of epithelial morphology and complete acquisition of mesenchymal morphology. Thus, upon acquiring partial EMT, cancer cell with E/M phenotypes can undergo collective cell migration through their remaining epithelial character and enhance attachment to the ECM by achieving mesenchymal character. Therefore, it was recently recognized that the ability of cancer cells to undergo partial EMT (E/M phenotype), rather than complete EMT, poses a higher metastatic risk. Notably, complete EMT is observed among cancer cells only under in vitro culture conditions. However, the possibility that the invading cancer cells have undergone complete EMT, and passive conversion to CTCs, due to release from the reactive stroma (see below), cannot be ruled out because it may not be possible to detect complete EMT among invading cells in vivo. Single or Collective Cancer Cell Migration Metastases, rather than the primary tumours that proliferate malignantly, are culpable for human cancer-associated mortality. Cancer cell invasion, during metastatic dissemination, can employ various interconvertible migration processes, collective cancer cell migration or single cancer cell migration. Collective cancer cell migration is characterized by multicellular clusters analogous to a bunch of grapes, and depends on cell–cell adhesion and multicellular coordination via cancer cells that express epithelial adhesion molecules. Single cancer cell migration, also known as mesenchymal migration, is characterized by fibroblast-like morphology, and depends on integrin-mediated interactions between cells and ECM (19). Pathologically, it is well-known that repression of E-cadherin, one of most characteristic proteins of EMT, is observed in cancer cells at the edges of tumour nests and at the invasion front of multicellular clusters, whereas E-cadherin is almost normally expressed in cancer cells at the centre of tumour nests of solid tumours (Fig. 1), thereby generating epigenetic heterogeneity (20). The molecular mechanism by which EMT occurs only in cancer cells at the edges of tumour nests and at the invasion front of multicellular clusters remains unanswered. EMT is regulated by ECM components, hypoxic conditions, exosomes and soluble factors such as hepatocyte growth factor (HGF), fibroblast growth factor (FGF) and members of transforming growth factor-βs (TGF-βs) (21). TGF-β was first described as an inducer of EMT during development (22). TGF-β is frequently overexpressed in cancer tissues, whereas cancer cells localize only to the invasion front and not to the centre of tumour nests, where they undergo EMT. This observation suggests that TGF-β alone is not sufficient to induce EMT in cancer cells, and that stromal tissues that neighbour cancer cells (known as the tumour microenvironment) are also required for EMT induction. The tumour microenvironment is composed of a variety of stromal cells including encompassing fibroblasts, inflammatory cells and endothelial cells in a dense collagenous ECM. Upon activating and co-opting the cellular components of the tumour microenvironment by ‘education signals’ from cancer cells, they can diffusely infiltrate the growing cancer and promote invasion by cancer cells by secreting a wide range of soluble factors (known as ‘re-education signals’). This desmoplastic microenvironment, sometimes referred to as reactive stroma, is composed of cancer-associated fibroblasts (CAFs), myofibroblasts and tumour-associated macrophages (TAMs), developing in conjunction with cancer, and behaves as a tumour-promoting driver (23). FGFs, HGF and tumour necrosis factor (TNF)-α, which are abundant in reactive stroma, induce the EMT of cancer cells in cooperation with TGF-β (24, 25; see below). Thus, cancer cells at the edges of tumour nests and at the invasion front of multicellular clusters, which are constitutively exposed to TGF-β secreted by cancer cells themselves, can respond to re-education signals presented in reactive stroma and undergo EMT, possibly leading to collective cancer cell migration. Fig. 1 View largeDownload slide E-cadherin repression at the invasion front of tumour. E-cadherin is expressed on the plasma membranes of cells at the centre of the tumour nest (arrow head), whereas its expression is substantially decreased or more diffuse in cells at the invasion front of the tumour and no longer localized to the plasma membranes (arrow). Fig. 1 View largeDownload slide E-cadherin repression at the invasion front of tumour. E-cadherin is expressed on the plasma membranes of cells at the centre of the tumour nest (arrow head), whereas its expression is substantially decreased or more diffuse in cells at the invasion front of the tumour and no longer localized to the plasma membranes (arrow). On the other hand, active Ras mutations are sometimes detected in various cancers, especially in pancreatic cancer (26). During the early stages of carcinogenesis, active Ras signals lead to the sustained activation of MAPK and reportedly help overcome the tumour suppressor effects of TGF-β, namely, growth inhibition and apoptosis induction, by promoting Smad degradation (27). Indeed, Ras transformation in epithelial cells belonging to various tissues confers resistance to growth inhibition by TGF-β. Thus, when Ras is actively mutated in epithelial cells and overcomes cell competition (28), these cells proliferate more rapidly in part by inhibiting TGF-β signals, leading to carcinogenesis. The population of cancer cells with Ras mutations expands rapidly and is gradually exposed to a large amount of TGF-β because Ras signals promote its autonomous secretion from cancer cells themselves (29). Consequently, Ras signals in cancer cells where TGF-β signalling molecules are intact can cooperate with TGF-β signals to induce EMT (see below) and promote efficient invasion into stromal tissues, possibly leading to single cancer cell migration (30). Fibroblast-Led Collective Cancer Cell Migration/Invasion CAFs promote tumour proliferation as well as tumour invasion and metastasis. Collective cancer cell migration by CAF, so-called fibroblast-led collective cell migration/invasion, was first described in 2007 (31). During such migration/invasion, carcinoma cells always move within tracks in the ECM behind the leading CAF. These CAF-generated tracks enable collective cancer cell migration through protease- and force-mediated matrix remodelling (31). In addition, the fibroblast-led collective cell migration is also supported by showing that the leading CAF realigns the forward ECM because the anterior expresses membrane-type 1 metalloprotease (MT1-MMP) to make single-cell caliber microtracks, and the following collective cells expand such narrow microtracks through the large-scale degradation of lateral ECM interfaces, ultimately prompting the transition towards collective cell migration (Fig. 2) (32). Recently, a heterophilic adherens junction involving E-cadherin on the cancer cell membrane and N-cadherin on the CAF membrane enables fibroblast-led collective cancer cell migration (Fig. 2) (33). Since cancer cells undergoing partial EMT (E/M phenotypes) localize to the interface between tumour nest and tumour stroma, it is possible that the interaction between cancer cells and CAFs via a heterophilic adherens junction is bridged by cancer cells undergoing partial EMT through a haemophilic adherens junction (Fig. 2). In addition, cancer cells undergoing EMT exhibit increased secretion of sonic hedgehog (SHH), but not desert hedgehog and Indian hedgehog, and induce increased metastasis by epithelial-like cancer cells with weakly metastatic properties by activating the Gli1 transcription factor (Fig. 2) (34). Therefore, heterotypic interactions among non-EMT cells and either EMT cells or CAF promote metastasis progression in various kinds of cancers. Fig. 2 View largeDownload slide Single or collective cancer cell migration. Single cancer cell migration, also known as mesenchymal migration, is characterized by fibroblast-like morphology (upper panel). Collective cancer cell migration is characterized by multicellular clusters analogous to a bunch of grapes, and includes fibroblast-led collective cancer cell migration/invasion (31), promotion of invasion by non-EMT cancer cells through crosstalk between EMT and non-EMT cancer cells (34), and putative EMT cell-mediated collective cancer cell migration. CAFs, non-EMT cancer cells and EMT cancer cells are shown in blue, pink and orange, respectively. N, N-cadherin; E, E-cadherin; SHH, sonic hedgehog; Gli1, zinc finger protein Gli1 (also known as glioma-associated oncogene). Fig. 2 View largeDownload slide Single or collective cancer cell migration. Single cancer cell migration, also known as mesenchymal migration, is characterized by fibroblast-like morphology (upper panel). Collective cancer cell migration is characterized by multicellular clusters analogous to a bunch of grapes, and includes fibroblast-led collective cancer cell migration/invasion (31), promotion of invasion by non-EMT cancer cells through crosstalk between EMT and non-EMT cancer cells (34), and putative EMT cell-mediated collective cancer cell migration. CAFs, non-EMT cancer cells and EMT cancer cells are shown in blue, pink and orange, respectively. N, N-cadherin; E, E-cadherin; SHH, sonic hedgehog; Gli1, zinc finger protein Gli1 (also known as glioma-associated oncogene). Mechanisms of Promoting Effects of FGF2 and Ras on EMT As described above that FGF2 and FGF4 enhance EMT (25), the underlying mechanism is quite unique. FGF receptor genes encode four functional receptors (FGFR1–FGFR4) that consist of three extracellular immunoglobulin-like domains (Ig-I, Ig-II and Ig-III), a transmembrane domain and an intracellular tyrosine kinase domain. The Ig-III domain is regulated by alternative splicing, which produces either the IIIb (FGFR1IIIb–FGFR3IIIb) or IIIc (FGFR1IIIc–FGFR3IIIc) isoforms that have distinct FGF binding specificities. Epithelial cells generally express the IIIb isoform and consequently respond to FGF-7, also known as KGF, and FGF-10. By contrast, mesenchymal cells express the IIIc isoform and respond to FGF-4 and FGF-2, also known as basic FGFs (35). Epithelial cells that undergo EMT by exposing TGF-β and aggressive cancer cells become sensitive to FGF-2 and FGF-4 through FGFRIIIc expression via the alternative splicing machinery (see below). Thus, FGF-2 and FGF-4 enhance EMT and sustain aggressive phenotypes (25, 36). Interestingly, cancer cells that become resistant to the anti-cancer drug trametinib, a MEK inhibitor, regain the ability to proliferate via reactivation of FGF-2 signals (37). The molecular mechanism by which Ras promotes EMT has been partially elucidated. Ras signals suppress TGF-β signals via phosphorylation of the linker domain of Smads, and facilitate inhibition of their translocation while promoting their degradation (30). Thus when the putative phosphorylation sites in the linker region of Smads are mutated, the resultant mutants become activated, compared to wild-type Smads (38). On the other hand, the resultant mutants activate EMT (Snail induction) to an extent similar to wild-type Smads, suggesting that phosphorylation of the linker domain in Smads is indispensable for EMT induction by Ras in collaboration with TGF-β (30). By screening an siRNA library targeting transcription factors, signal transducer and activator of transcription 3 (a.k.a. STAT3) was identified as a mediator that synergizes the Ras and TGF-β signals (39). The detailed underlying mechanism remains unclear, but STAT3 is partly involved in EMT induction by TGF-β in cooperation with Ras signals (Fig. 3). Fig. 3 View largeDownload slide Synergism between TGF-β and Ras signals for EMT induction. RasG12V enhances TGF-β–induced Snail and EMT, whereas it suppresses TGF-β–induced growth inhibition. STAT3 mediates TGF-β–induced EMT (39). Fig. 3 View largeDownload slide Synergism between TGF-β and Ras signals for EMT induction. RasG12V enhances TGF-β–induced Snail and EMT, whereas it suppresses TGF-β–induced growth inhibition. STAT3 mediates TGF-β–induced EMT (39). Detection of Cancer Cells Undergoing EMT It is becoming increasingly clear that partial EMT, rather than complete EMT, is important for cancer progression, but not for development (8). Before proposing this concept, we sought to identify marker proteins, which discriminate cancer cells undergoing EMT from fibroblasts and CAF in the tumour microenvironment. By microarray analyses using cells treated with FGF-2 in combination with TGF-β in vitro, we identified three molecules, namely, RGS16, PAI-2 and ITGA3. All three proteins are upregulated by this combination, but not by either alone, although expression levels of RGS16 do not correspond completely with aggressive phenotypes of breast cancer cells (40). At around the same time, PAI-2, also known as SERPIN2, was identified as a molecule that promotes the survival and brain metastasis of lung and breast cancer cells (41). Expression of ITGA3, that is integrin α3, correlated positively with aggressive phenotypes of breast cancer cells at mRNA and protein levels, and was not detected in several established fibroblasts in vitro. Immunohistochemical analyses using human specimens show that ITGA3 is clearly stained in cancer cells, but faintly in fibroblasts or certain cells in the tumour microenvironment (42). Therefore, these molecules identified from the expression screening using microarray analyses are useful for detecting cancer cells with more aggressive phenotypes, but not sufficient to specifically detect only cancer cells undergoing EMT. Based on changes in alternative splicing during EMT, research into specific markers to detect cancer cells undergoing EMT is in progress. As described above, the alternative splicing of FGFR-encoding transcripts occurs during EMT and is caused by ESRP1 and ESRP2. ESRPs bind directly to alternatively spliced regions of transcripts that encode FGFR, CD44, Rac1, p120 catenin, and Mena (mammalian Ena, a member of the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) family of proteins) (Fig. 4) (36, 43). Although the functions of different ESRPs differ slightly from each other, ESRPs are highly expressed in epithelial cells but strongly downregulated in cells undergoing EMT (44). In fact, immunohistochemical analyses shows that ESRP expression levels are low in normal epithelium, upregulated in precancerous lesions and carcinoma in situ, maintained in advanced cancer cells, and finally, downregulated in cells at invasive fronts (44). Although ESRPs are the major regulators of controlling epithelial-specific splicing, RNA-binding motif protein 47 (RBM47) is also downregulated during EMT and regulates numerous transcripts that undergo a switch in alternative splicing during EMT in cooperation with ESRPs (45). Besides epithelial-specific splicing, mesenchymal-specific splicing is mediated by RBFOX2, MBNL1 and SRSF1 during EMT. Fig. 4 View largeDownload slide Schematic illustrations of EMT regulated by transcriptional and post-transcriptional mechanisms. EMT transcriptional factors (EMT-TFs) decrease expression of ESRPs, leading to changes in alternative splicing events. FSP, fibroblast-specific protein; SMA, smooth muscle α-actin; FGFR, fibroblast growth factor receptor; RON, a scatter factor receptor and proto-oncogene (also known as MST1R); p120ctn, p120 catenin; ENAH, a member of the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) family of proteins; CPEB, cytosolic polyadenylation element–binding protein 2. Fig. 4 View largeDownload slide Schematic illustrations of EMT regulated by transcriptional and post-transcriptional mechanisms. EMT transcriptional factors (EMT-TFs) decrease expression of ESRPs, leading to changes in alternative splicing events. FSP, fibroblast-specific protein; SMA, smooth muscle α-actin; FGFR, fibroblast growth factor receptor; RON, a scatter factor receptor and proto-oncogene (also known as MST1R); p120ctn, p120 catenin; ENAH, a member of the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) family of proteins; CPEB, cytosolic polyadenylation element–binding protein 2. ΔRon, a constitutively active isoform of the Ron tyrosine kinase receptor, is produced when Ron exon 11 is skipped. This skipping is induced by SRSF1 during EMT, but inhibited by hnRNPA1 through antagonizing the binding of SRSF1 (46, 47). Rac1b, a constitutively active isoform of Rac1, is generated by the inclusion of exon 3b, which is enhanced by SRSF1 and antagonized by SRSF3 during EMT (48). Alternative splicing of Rac1b is also regulated by ESRPs (44). CD44 splicing toward the CD44s isoform is regulated by ESRPs, which are downregulated during EMT, whereas hnRNPM inhibits it (36, 49). In addition, the splicing of CD44v to CD44s during EMT is regulated through T179 phosphorylation of Smad3-mediated interaction with hnRNPE1, also known as PCBP1 (50). Similar to ESRPs, splicing of FGFRIIIc to IIIb is also regulated by KHSRP in association with hnRNPA1 (51). However, molecules responsible for converting FGFRIIIb to IIIc have not been fully elucidated to date. Taken together, gene products generated by alternative splicing could be useful as potential molecular markers for diagnosis for cancer cells undergoing EMT and aggressive cancer cells. More interestingly, oncogenic Ras collaborates with TGF-β to induce EMT in cancer cells (30). Although the change in alternative splicing during EMT induced by oncogenic Ras and TGF-β has yet to be elucidated, the gene products generated from oncogenic Ras-mediated alternative splicing could differ from those produced during developmental EMT and therefore be useful as potential diagnostic molecular markers that specifically identify invading cancer cells that undergo EMT in the tumour microenvironment. EMT Transcription Factors (EMT-TFs) in Breast Cancer Cells Transcriptional repression of E-cadherin, which is frequently observed in malignant tumours, is mediated by EMT-TFs, including the δEF1 family of two-handed zinc-finger factors (ZEB1 [Zinc-finger E-box binding homeobox 1]/δEF1 [δ-crystallin/E2-box factor 1] and ZEB2/SIP1 [Smad-interacting protein1]), the Snai1 family (Snail, Slug and Smuc) and basic helix-loop-helix factors (Twist and E12/E47) (21). Among these, the levels of δEF1 family proteins (ZEB1/δEF1 and ZEB2/SIP1) in particular correlate positively with EMT phenotypes and aggressiveness of breast cancer cells (36, 52) (Fig. 5). Molecular profiling of breast cancers revealed two distinct molecular subtypes in which gene expression patterns agree precisely with pathological and clinical features. These subtypes, luminal and basal-like, are generally thought to be composed exclusively of cells with epithelial and mesenchymal phenotypes, respectively (53). Fig. 5 View largeDownload slide Expression profiles of E-cadherin, and the EMT regulators in breast cancer cells. mRNA levels of the expression of E-cadherin, and the EMT regulators were compared among 23 human breast cancer cell lines by quantitative RT-PCR (36, 52). Gene cluster shown is reported by Neve et al. (53). *, Luminal subtype; *, basal-like subtype. Fig. 5 View largeDownload slide Expression profiles of E-cadherin, and the EMT regulators in breast cancer cells. mRNA levels of the expression of E-cadherin, and the EMT regulators were compared among 23 human breast cancer cell lines by quantitative RT-PCR (36, 52). Gene cluster shown is reported by Neve et al. (53). *, Luminal subtype; *, basal-like subtype. The basal-like subtype, associated with aggressive behaviour and poor prognosis, shows low levels of E-cadherin and ESRPs, and high levels of ZEBs, ITGA3, p-ERK and NF-kB proteins, whereas the luminal subtype shows opposite expression profiles of these proteins (36, 42, 54). Interestingly, Ets1, one member of the E26 transformation-specific (ETS) family of transcription factors, is highly expressed in the basal-like subtype, whereas ESE1, a member of the Ets transcription factor family, is highly expressed in the luminal subtype, and repressed ZEBs through Ets1 repression (55). Moreover, levels of E-cadherin are also sometimes accompanied by hypermethylation of the promoter region (55). In aggressive cancer cells such as basal-like subtypes, ZEBs recruit DNMT1 to hemi-methylated DNA in the promoter region of E-cadherin, resulting in reduced expression of E-cadherin via hypermethylation (52). Thus, ZEBs acts as a transcriptional repressor to directly suppress E-cadherin expression, and as a potent epigenetic regulator (in cooperation with DNMT1) to maintain E-cadherin repression. Additionally, post-translational modifications, such as phosphorylation, ubiquitination and sumoylation, are largely known to regulate EMT-TFs. Moreover, several non-coding RNAs, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have been shown to play a role in EMT through regulating expression of EMT-TFs (2, 21, 56). A recently proposed model known as the ‘ceRNA theory’ and the nonsense-mediated mRNA decay pathway may be involved in EMT induction through multiple gene-expression networks, including EMT-TFs, in cancer cells (57). Therefore, the EMT-TFs that are regulated at both the transcriptional and post-translational levels modify expressions of EMT marker proteins at the transcriptional, post-transcriptional and epigenetic levels, and regulate EMT and aggressiveness in cooperation with additional factors secreted from cells and/or the cellular components in the tumour microenvironment. Perspectives During EMT in cancer progression, cancer cells exhibit both mesenchymal and epithelial characteristics, known as partial EMT. Recently, it was recognized that the ability of cancer cells to undergo partial EMT, rather than complete EMT, poses a higher metastatic risk. Moreover, partial EMT states can be associated with cancer stem/progenitor cell functions, thus generating various cellular phenotypes through self-renewal or asymmetric divisions. However, molecular mechanisms how partial EMT is regulated are still unclear. Thus, as-yet-unidentified molecules that determine the partial EMT represent promising targets for both the diagnosis and treatment of cancer. Acknowledgements I thank Dr. K. Sakamoto for valuable discussion and all members of department of Biochemistry of University of Yamanashi for consistently supporting this study. Funding This work was supported by THE MITSUBISHI FOUNDATION and JSPS KAKENHI Grant Number JP15H05018. Conflict of Interest None declared. References 1 Greenburg G. , Hay E.D. ( 1982 ) Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells . J. Cell Biol . 95 , 333 – 339 Google Scholar CrossRef Search ADS PubMed 2 Nieto M.A. , Huang R.Y. , Jackson R.A. , Thiery J.P. ( 2016 ) Emt: 2016 . Cell 166 , 21 – 45 Google Scholar CrossRef Search ADS PubMed 3 Nawshad A. , LaGamba D. , Hay E.D. ( 2004 ) Transforming growth factor β (TGFβ) signalling in palatal growth, apoptosis and epithelial mesenchymal transformation (EMT) . Arch. Oral Biol . 49 , 675 – 689 Google Scholar CrossRef Search ADS PubMed 4 Martı´nez-Álvarez C. , Blanco M.J. , Pérez R. , Rabadán M.A. , Aparicio M. , Resel E. , Martı´nez T. , Nieto M.A. , ( 2004 ) Snail family members and cell survival in physiological and pathological cleft palates . Dev. Biol . 265 , 207 – 218 Google Scholar CrossRef Search ADS PubMed 5 Takigawa T. , Shiota K. ( 2004 ) Terminal differentiation of palatal medial edge epithelial cells in vitro is not necessarily dependent on palatal shelf contact and midline epithelial seam formation . Int. J. Dev. Biol. 48 , 307 – 317 Google Scholar CrossRef Search ADS PubMed 6 Hay E.D. , Zuk A. ( 1995 ) Transformations between epithelium and mesenchyme: normal, pathological, and experimentally induced . Am. J. Kidney Dis . 26 , 678 – 690 Google Scholar CrossRef Search ADS PubMed 7 Kalluri R. ( 2009 ) EMT: when epithelial cells decide to become mesenchymal-like cells . J. Clin. Invest. 119 , 1417 – 1419 Google Scholar CrossRef Search ADS PubMed 8 Kalluri R. , Weinberg R.A. ( 2009 ) The basics of epithelial-mesenchymal transition . J. Clin. Invest. 119 , 1420 – 1428 Google Scholar CrossRef Search ADS PubMed 9 Haensel D. , Dai X. ( 2018 ) Epithelial-to-mesenchymal transition in cutaneous wound healing: where we are and where we are heading . Dev. Dyn. 247 , 473 – 480 Google Scholar CrossRef Search ADS PubMed 10 Zeisberg M. , Kalluri R. ( 2004 ) The role of epithelial-to-mesenchymal transition in renal fibrosis . J. Mol. Med. 82 , 175 – 181 Google Scholar CrossRef Search ADS PubMed 11 Thiery J.P. , Acloque H. , Huang R.Y. , Nieto M.A. ( 2009 ) Epithelial-mesenchymal transitions in development and disease . Cell 139 , 871 – 890 Google Scholar CrossRef Search ADS PubMed 12 Geiger T.R. , Peeper D.S. ( 2009 ) Metastasis mechanisms . Biochim. Biophys. Acta 1796 , 293 – 308 Google Scholar PubMed 13 Scheel C. , Weinberg R.A. ( 2012 ) Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links . Semin. Cancer Biol . 22 , 396 – 403 Google Scholar CrossRef Search ADS PubMed 14 Jie X.X. , Zhang X.Y. , Xu C.J. ( 2017 ) Epithelial-to-mesenchymal transition, circulating tumor cells and cancer metastasis: mechanisms and clinical applications . Oncotarget 8 , 81558 – 81571 Google Scholar PubMed 15 Nieto M.A. ( 2017 ) Context-specific roles of EMT programmes in cancer cell dissemination . Nat. Cell Biol. 19 , 416 – 418 Google Scholar CrossRef Search ADS PubMed 16 Furuya S. , Endo K. , Takahashi A. , Miyazawa K. , Saitoh M. ( 2017 ) Snail suppresses cellular senescence and promotes fibroblast-led cancer cell invasion . FEBS Open Biol . 7 , 1586 – 1597 Google Scholar CrossRef Search ADS 17 Shibue T. , Weinberg R.A. ( 2017 ) EMT, CSCs, and drug resistance: the mechanistic link and clinical implications . Nat. Rev. Clin. Oncol. 14 , 611 – 629 Google Scholar CrossRef Search ADS PubMed 18 Yu M. , Bardia A. , Wittner B.S. , Stott S.L. , Smas M.E. , Ting D.T. , Isakoff S.J. , Ciciliano J.C. , Wells M.N. , Shah A.M. , Concannon K.F. , Donaldson M.C. , Sequist L.V. , Brachtel E. , Sgroi D. , Baselga J. , Ramaswamy S. , Toner M. , Haber D.A. , Maheswaran S. ( 2013 ) Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition . Science 339 , 580 – 584 Google Scholar CrossRef Search ADS PubMed 19 Lintz M. , Munoz A. , Reinhart-King C.A. ( 2017 ) The mechanics of single cell and collective migration of tumor cells . J. Biomech. Eng. 139 , 021005 Google Scholar CrossRef Search ADS 20 Shibata M. , Shen M.M. ( 2013 ) The roots of cancer: stem cells and the basis for tumor heterogeneity . BioEssays 35 , 253 – 260 Google Scholar CrossRef Search ADS PubMed 21 Saitoh M. ( 2015 ) Epithelial-mesenchymal transition is regulated at post-transcriptional levels by transforming growth factor-β signaling during tumor progression . Cancer Sci. 106 , 481 – 488 Google Scholar CrossRef Search ADS PubMed 22 Miettinen P.J. , Ebner R. , Lopez A.R. , Derynck R. ( 1994 ) TGF-β induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors . J. Cell Biol . 127 , 2021 – 2036 Google Scholar CrossRef Search ADS PubMed 23 Orimo A. , Gupta P.B. , Sgroi D.C. , Arenzana-Seisdedos F. , Delaunay T. , Naeem R. , Carey V.J. , Richardson A.L. , Weinberg R.A. ( 2005 ) Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion . Cell 121 , 335 – 348 Google Scholar CrossRef Search ADS PubMed 24 Kawata M. , Koinuma D. , Ogami T. , Umezawa K. , Iwata C. , Watabe T. , Miyazono K. ( 2012 ) TGF-β-induced epithelial-mesenchymal transition of A549 lung adenocarcinoma cells is enhanced by pro-inflammatory cytokines derived from RAW 264.7 macrophage cells . J. Biochem . 151 , 205 – 216 Google Scholar CrossRef Search ADS PubMed 25 Shirakihara T. , Horiguchi T. , Miyazawa M. , Ehata S. , Shibata T. , Morita I. , Miyazono K. , Saitoh M. ( 2011 ) TGF-β regulates isoform switching of FGF receptors and epithelial-mesenchymal transition . EMBO J . 30 , 783 – 795 Google Scholar CrossRef Search ADS PubMed 26 Zhou L. , Baba Y. , Kitano Y. , Miyake K. , Zhang X. , Yamamura K. , Kosumi K. , Kaida T. , Arima K. , Taki K. , Higashi T. , Imai K. , Hashimoto D. , Yamashita Y. , Chikamoto A. , Beppu T. , Tan X. , Baba H. ( 2016 ) KRAS, BRAF, and PIK3CA mutations, and patient prognosis in 126 pancreatic cancers: pyrosequencing technology and literature review . Med. Oncol . 33 , 32 Google Scholar CrossRef Search ADS PubMed 27 Kretzschmar M. , Doody J. , Timokhina I. , Massague J. ( 1999 ) A mechanism of repression of TGFbeta/Smad signaling by oncogenic Ras . Genes Dev. 13 , 804 – 816 Google Scholar CrossRef Search ADS PubMed 28 Kajita M. , Fujita Y. ( 2015 ) EDAC: epithelial defence against cancer-cell competition between normal and transformed epithelial cells in mammals . J. Biochem . 158 , 15 – 23 Google Scholar CrossRef Search ADS PubMed 29 Schwarz L.C. , Wright J.A. , Gingras M.C. , Kondaiah P. , Danielpour D. , Pimentel M. , Sporn M.B. , Greenberg A.H. ( 1990 ) Aberrant TGF-β production and regulation in metastatic malignancy . Growth Factors 3 , 115 – 127 Google Scholar CrossRef Search ADS PubMed 30 Horiguchi K. , Shirakihara T. , Nakano A. , Imamura T. , Miyazono K. , Saitoh M. ( 2009 ) Role of Ras signaling in the induction of snail by transforming growth factor-β . J. Biol. Chem. 284 , 245 – 253 Google Scholar CrossRef Search ADS PubMed 31 Gaggioli C. , Hooper S. , Hidalgo-Carcedo C. , Grosse R. , Marshall J.F. , Harrington K. , Sahai E. ( 2007 ) Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells . Nat. Cell Biol. 9 , 1392 – 1400 Google Scholar CrossRef Search ADS PubMed 32 Wolf K. , Wu Y.I. , Liu Y. , Geiger J. , Tam E. , Overall C. , Stack M.S. , Friedl P. ( 2007 ) Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion . Nat. Cell Biol. 9 , 893 – 904 Google Scholar CrossRef Search ADS PubMed 33 Labernadie A. , Kato T. , Brugues A. , Serra-Picamal X. , Derzsi S. , Arwert E. , Weston A. , Gonzalez-Tarrago V. , Elosegui-Artola A. , Albertazzi L. , Alcaraz J. , Roca-Cusachs P. , Sahai E. , Trepat X. ( 2017 ) A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion . Nat. Cell Biol. 19 , 224 – 237 Google Scholar CrossRef Search ADS PubMed 34 Neelakantan D. , Zhou H. , Oliphant M.U.J. , Zhang X. , Simon L.M. , Henke D.M. , Shaw C.A. , Wu M.F. , Hilsenbeck S.G. , White L.D. , Lewis M.T. , Ford H.L. ( 2017 ) EMT cells increase breast cancer metastasis via paracrine GLI activation in neighbouring tumour cells . Nat. Commun. 8 , 15773 Google Scholar CrossRef Search ADS PubMed 35 Eswarakumar V.P. , Lax I. , Schlessinger J. ( 2005 ) Cellular signaling by fibroblast growth factor receptors . Cytokine Growth Factor Rev . 16 , 139 – 149 Google Scholar CrossRef Search ADS PubMed 36 Horiguchi K. , Sakamoto K. , Koinuma D. , Semba K. , Inoue A. , Inoue S. , Fujii H. , Yamaguchi A. , Miyazawa K. , Miyazono K. , Saitoh M. ( 2012 ) TGF-β drives epithelial-mesenchymal transition through δEF1-mediated downregulation of ESRP . Oncogene 31 , 3190 – 3201 Google Scholar CrossRef Search ADS PubMed 37 Manchado E. , Weissmueller S. , Morris JPt. , Chen C.C. , Wullenkord R. , Lujambio A. , de Stanchina E. , Poirier J.T. , Gainor J.F. , Corcoran R.B. , Engelman J.A. , Rudin C.M. , Rosen N. , Lowe S.W. ( 2016 ) A combinatorial strategy for treating KRAS-mutant lung cancer . Nature 534 , 647 – 651 Google Scholar CrossRef Search ADS PubMed 38 Nakano A. , Koinuma D. , Miyazawa K. , Uchida T. , Saitoh M. , Kawabata M. , Hanai J. , Akiyama H. , Abe M. , Miyazono K. , Matsumoto T. , Imamura T. ( 2009 ) Pin1 down-regulates transforming growth factor-β (TGF-β) signaling by inducing degradation of Smad proteins . J. Biol. Chem. 284 , 6109 – 6115 Google Scholar CrossRef Search ADS PubMed 39 Saitoh M. , Endo K. , Furuya S. , Minami M. , Fukasawa A. , Imamura T. , Miyazawa K. ( 2016 ) STAT3 integrates cooperative Ras and TGF-β signals that induce Snail expression . Oncogene 35 , 1049 – 1057 Google Scholar CrossRef Search ADS PubMed 40 Hoshi Y. , Endo K. , Shirakihara T. , Fukagawa A. , Miyazawa K. , Saitoh M. ( 2016 ) The potential role of regulator of G-protein signaling 16 in cell motility mediated by δEF1 family proteins . FEBS Lett. 590 , 270 – 278 Google Scholar CrossRef Search ADS PubMed 41 Valiente M. , Obenauf A.C. , Jin X. , Chen Q. , Zhang X.H. , Lee D.J. , Chaft J.E. , Kris M.G. , Huse J.T. , Brogi E. , Massague J. ( 2014 ) Serpins promote cancer cell survival and vascular co-option in brain metastasis . Cell 156 , 1002 – 1016 Google Scholar CrossRef Search ADS PubMed 42 Shirakihara T. , Kawasaki T. , Fukagawa A. , Semba K. , Sakai R. , Miyazono K. , Miyazawa K. , Saitoh M. ( 2013 ) Identification of integrin α3 as a molecular marker of cells undergoing epithelial-mesenchymal transition and of cancer cells with aggressive phenotypes . Cancer Sci. 104 , 1189 – 1197 Google Scholar CrossRef Search ADS PubMed 43 Warzecha C.C. , Sato T.K. , Nabet B. , Hogenesch J.B. , Carstens R.P. ( 2009 ) ESRP1 and ESRP2 are epithelial cell-type-specific regulators of FGFR2 splicing . Mol. Cell. 33 , 591 – 601 Google Scholar CrossRef Search ADS PubMed 44 Ishii H. , Saitoh M. , Sakamoto K. , Kondo T. , Katoh R. , Tanaka S. , Motizuki M. , Masuyama K. , Miyazawa K. ( 2014 ) Epithelial splicing regulatory proteins 1 (ESRP1) and 2 (ESRP2) suppress cancer cell motility via different mechanisms . J. Biol. Chem. 289 , 27386 – 27399 Google Scholar CrossRef Search ADS PubMed 45 Yang Y. , Park J.W. , Bebee T.W. , Warzecha C.C. , Guo Y. , Shang X. , Xing Y. , Carstens R.P. ( 2016 ) Determination of a comprehensive alternative splicing regulatory network and combinatorial regulation by key factors during the epithelial-to-mesenchymal transition . Mol. Cell. Biol. 36 , 1704 – 1719 Google Scholar CrossRef Search ADS PubMed 46 Mayer S. , Hirschfeld M. , Jaeger M. , Pies S. , Iborra S. , Erbes T. , Stickeler E. ( 2015 ) RON alternative splicing regulation in primary ovarian cancer . Oncol. Rep . 34 , 423 – 430 Google Scholar CrossRef Search ADS PubMed 47 Bonomi S. , di Matteo A. , Buratti E. , Cabianca D.S. , Baralle F.E. , Ghigna C. , Biamonti G. ( 2013 ) HnRNP A1 controls a splicing regulatory circuit promoting mesenchymal-to-epithelial transition . Nucleic Acids Res . 41 , 8665 – 8679 Google Scholar CrossRef Search ADS PubMed 48 Goncalves V. , Henriques A.F. , Pereira J.F. , Neves Costa A. , Moyer M.P. , Moita L.F. , Gama-Carvalho M. , Matos P. , Jordan P. ( 2014 ) Phosphorylation of SRSF1 by SRPK1 regulates alternative splicing of tumor-related Rac1b in colorectal cells . RNA 20 , 474 – 482 Google Scholar CrossRef Search ADS PubMed 49 Xu Y. , Gao X.D. , Lee J.H. , Huang H. , Tan H. , Ahn J. , Reinke L.M. , Peter M.E. , Feng Y. , Gius D. , Siziopikou K.P. , Peng J. , Xiao X. , Cheng C. ( 2014 ) Cell type-restricted activity of hnRNPM promotes breast cancer metastasis via regulating alternative splicing . Genes Dev . 28 , 1191 – 1203 Google Scholar CrossRef Search ADS PubMed 50 Tripathi V. , Sixt K.M. , Gao S. , Xu X. , Huang J. , Weigert R. , Zhou M. , Zhang Y.E. ( 2016 ) Direct regulation of alternative splicing by SMAD3 through PCBP1 is essential to the tumor-promoting role of TGF-β . Mol. Cell 64 , 1010 Google Scholar CrossRef Search ADS PubMed 51 Puppo M. , Bucci G. , Rossi M. , Giovarelli M. , Bordo D. , Moshiri A. , Gorlero F. , Gherzi R. , Briata P. ( 2016 ) miRNA-mediated KHSRP silencing rewires distinct post-transcriptional programs during TGF-β-induced epithelial-to-mesenchymal transition . Cell Rep . 16 , 967 – 978 Google Scholar CrossRef Search ADS PubMed 52 Fukagawa A. , Ishii H. , Miyazawa K. , Saitoh M. ( 2015 ) δEF1 associates with DNMT1 and maintains DNA methylation of the E-cadherin promoter in breast cancer cells . Cancer Med. 4 , 125 – 135 Google Scholar CrossRef Search ADS PubMed 53 Neve R.M. , Chin K. , Fridlyand J. , Yeh J. , Baehner F.L. , Fevr T. , Clark L. , Bayani N. , Coppe J.P. , Tong F. , Speed T. , Spellman P.T. , DeVries S. , Lapuk A. , Wang N.J. , Kuo W.L. , Stilwell J.L. , Pinkel D. , Albertson D.G. , Waldman F.M. , McCormick F. , Dickson R.B. , Johnson M.D. , Lippman M. , Ethier S. , Gazdar A. , Gray J.W. ( 2006 ) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes . Cancer Cell 10 , 515 – 527 Google Scholar CrossRef Search ADS PubMed 54 Yamaguchi N. , Ito T. , Azuma S. , Ito E. , Honma R. , Yanagisawa Y. , Nishikawa A. , Kawamura M. , Imai J. , Watanabe S. , Semba K. , Inoue J. ( 2009 ) Constitutive activation of nuclear factor-κB is preferentially involved in the proliferation of basal-like subtype breast cancer cell lines . Cancer Sci . 100 , 1668 – 1674 Google Scholar CrossRef Search ADS PubMed 55 Sinh N.D. , Endo K. , Miyazawa K. , Saitoh M. ( 2017 ) Ets1 and ESE1 reciprocally regulate expression of ZEB1/ZEB2, dependent on ERK1/2 activity, in breast cancer cells . Cancer Sci. 108 , 952 – 960 Google Scholar CrossRef Search ADS PubMed 56 Yamamoto M. , Taguchi Y. , Ito-Kureha T. , Semba K. , Yamaguchi N. , Inoue J. ( 2013 ) NF-κB non-cell-autonomously regulates cancer stem cell populations in the basal-like breast cancer subtype . Nat. Commun . 4 , 2299 Google Scholar CrossRef Search ADS PubMed 57 Tay Y. , Rinn J. , Pandolfi P.P. ( 2014 ) The multilayered complexity of ceRNA crosstalk and competition . Nature 505 , 344 – 352 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations CAF cancer-associated fibroblasts CSC cancer stem cell CTC circulating tumour cells EMT epithelial–mesenchymal transition MET mesenchymal–epithelial transition TGF-β transforming growth factor-βs © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
The Journal of Biochemistry – Oxford University Press
Published: May 3, 2018
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera