Epithelial-Mesenchymal Transition in Cancer: Parallels Between Normal Development and Tumor Progression
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From the earliest stages of embryonic development, cells of epithelial and mesenchymal origin contribute to the structure and function of developing organs. However, these phenotypes are not always permanent, and instead, under the appropriate conditions, epithelial and mesenchymal cells convert between these two phenotypes. These processes, termed Epithelial-Mesenchymal Transition (EMT), or the reverse Mesenchymal-Epithelial Transition (MET), are required for complex body patterning and morphogenesis. In addition, epithelial plasticity and the acquisition of invasive properties without the full commitment to a mesenchymal phenotype are critical in development, particularly during branching morphogenesis in the mammary gland. Recent work in cancer has identified an analogous plasticity of cellular phenotypes whereby epithelial cancer cells acquire mesenchymal features that permit escape from the primary tumor. Because local invasion is thought to be a necessary first step in metastatic dissemination, EMT and epithelial plasticity are hypothesized to contribute to tumor progression. Similarities between developmental and oncogenic EMT have led to the identification of common contributing pathways, suggesting that the reactivation of developmental pathways in breast and other cancers contributes to tumor progression. For example, developmental EMT regulators including Snail/Slug, Twist, Six1, and Cripto, along with developmental signaling pathways including TGF-β and Wnt/β-catenin, are misexpressed in breast cancer and correlate with poor clinical outcomes. This review focuses on the parallels between epithelial plasticity/EMT in the mammary gland and other organs during development, and on a selection of developmental EMT regulators that are misexpressed specifically during breast cancer.
KeywordsEpithelial-Mesenchymal transition Epithelial plasticity Breast cancer metastasis Branching morphogenesis
Transforming Growth Factor-β
Fibroblast Growth Factor
Bone Morphogenetic Protein
Medial Edge Epithelium
Terminal End Bud
Epidermal Growth Factor
Hepatocyte Growth Factor/Scatter Factor
Whey Acidic Protein
Mouse Mammary Tumor Virus
The evolution of multicellular organisms permitted the development of specialized cell types and the diversification of cellular phenotypes. One of the most primitive divergences in cell phenotype in early organisms is the distinction between epithelial and mesenchymal cells. Epithelial cells provide cell-cell cohesion essential to maintaining the integrity of the multicellular organism and function as a critical barrier necessary for establishing a regulated internal environment, independent from the external environment . In mammals, epithelialization of the developing embryo occurs early during compaction of the blastula [2, 3]. However, the development of more complex body structures and functions requires the flexibility afforded by another cell type, the mesenchymal cell. Shortly after the epithelialization of the blastula, the primary mesenchyme is formed during gastrulation, providing the first distinction between epithelial and mesenchymal phenotypes . Cells exhibiting a mesenchymal phenotype provide support and structure to the epithelial cells particularly through the production of an extracellular matrix and, unlike the rather confined and immobile epithelial cell, are highly motile and invasive . From the evolution of primitive trilaminar body patterns to the complex development and organogenesis of mammals, epithelial and mesenchymal cell phenotypes are a basic feature of normal development and physiology. However, depending on the cell type and its particular environment, epithelial and mesenchymal cell phenotypes are not static and instead can be highly dynamic. Interconversion between epithelial and mesenchymal cell phenotypes, processes termed Epithelial-Mesenchymal Transition (EMT) and the reverse Mesenchymal-Epithelial Transition (MET), provides additional flexibility particularly during embryogenesis, but also permits dynamic cellular remodeling during wound healing and regeneration of fully differentiated tissues [1, 6].
EMT, and the reverse process MET, have both been thoroughly studied in mammalian development, where numerous embryonic events and developing organs depend on the switch between epithelial and mesenchymal phenotypes including gastrulation , neural crest formation , palatogenesis , heart valve formation , nephrogenesis  and myogenesis . While EMT is a critical normal process during development and wound healing, recently properties of EMT have been implicated in human pathology, including fibrosis and cancer metastasis . Not surprisingly, many of the same signaling pathways and transcription factors important to physiologic instances of EMT are also activated during pathologic EMT. While in the adult, a quiescent epithelium does not typically exhibit features of EMT. However, in the right context, either due to an injurious insult or the genetic and environmental perturbations of cancer, the epithelium may become activated and primed for induction of epithelial plasticity and EMT. Particularly in cancer, parallels with normal development have been well established [13, 14]. The implication of this observation is that cancer cells may readily reactivate developmental properties out of context in the adult, which then contribute to tumorigenesis via inducing accelerated proliferation, resistance to apoptosis and evasion of senescence. This idea extends beyond tumor formation; the reactivation of developmental programs such as EMT could facilitate tumor cell invasion, spread and metastasis, and therefore may also represent a major mechanism of tumor progression. Consistent with this hypothesis, numerous signaling pathways and transcription factors identified as critical mediators of developmental EMT have also been implicated in oncogenic EMT and in tumor progression.
Interestingly, the induction of a complete EMT is not the only mechanism by which epithelial cells can migrate and spread during development. In addition to EMT, epithelial cell plasticity has also been observed in a process termed collective migration. This process entails the movement of epithelial cells as a group that is both physically and functionally connected . Collective migration has been identified as a critical cellular process particularly during mammary gland branching morphogenesis , intestinal epithelial differentiation  and wound healing . Although during collective migration epithelial cells maintain cell-cell junctions, many properties of collective migration parallel changes observed during EMT, including alteration of apico-basal polarity, modification of the extracellular matrix and the acquisition of an invasive and motile phenotype . The similarities between EMT and collective migration suggest that the transition between epithelial and mesenchymal cell phenotypes may not always represent two absolute and independent cellular states, but instead a continuous spectrum of epithelial and mesenchymal properties. This theory is particularly attractive in the context of cancer, which typically lacks the coordinated and orderly induction of a complete EMT. Instead, in cancer, the highly variable environmental cues coupled with genetic heterogeneity of the tumor may lead to varying degrees of epithelial plasticity and reactivation of developmental migratory programs, resulting in the induction of single cell and/or collective cell migration. In this review, we will discuss EMT and epithelial plasticity in development, as well as the evidence for reactivation of these developmental programs in cancer progression and metastasis. We will then focus on select examples of developmental EMT regulators which have been identified as critical contributors to breast cancer progression specifically.
Defining Epithelial-Mesenchymal Transition (EMT) in Development and in Cancer
During development, the steps of EMT are well-defined and typically accompanied by cell fate decisions, however the EMT associated with cancer and fibrosis is less clearly defined and specific to each pathology. While the basic features of EMT are similar among the different conditions, a recent series of reviews has proposed classifying EMT into three different subtypes, based on the functional consequences and biological context, in an attempt to better reflect the distinctive properties of EMT in development. These three types are developmental (Type I), fibrosis and wound healing (Type II), and cancer (Type III) . In addition, biomarkers specific to each subtype of EMT and also common to all subtypes have been defined . Overall, EMT is characterized as a downregulation of epithelial markers, particularly E-cadherin, and an upregulation of mesenchymal markers, particularly vimentin or fibronectin, accompanied by an increase in cell migration and invasion.
Examples of Developmental EMT
As development progresses, another example of EMT occurs in cells of the neuroectoderm that generate a second population of primary mesenchymal cells as the neural tube closes (Fig. 3b). As neurulation occurs in vertebrate development, cells near the junction of the neuroectoderm and the embryonic ectoderm undergo EMT, generating a migratory neural crest cell population . After delamination and migration throughout the developing embryo, these cells give rise to various cell types and tissue structures including facial and cervical structures, the neurons of the peripheral nervous system, pigment cells and the cells of the adrenal medulla. The molecular basis of neural crest EMT includes expression of the E-cadherin repressor, Snail . Induction of Snail likely is under control of converging signals including Bone Morphogenetic Protein (BMP), Nodal, Wnt and FGFs . An interesting aspect of neural crest migration revolves around the extensive cell-cell interactions that occur after EMT and the delamination from the ectoderm. In fact, the migration of neural crest cells has been described as collective migration, since the mesenchymal cells transiently interact through both physical and biochemical interactions, thereby modulating neighboring cell migration . This interaction between neighboring neural crest cells allows them to move in close proximity as an organized stream.
In addition to gastrulation and neural crest formation, EMT is also critical in the development of other organs including the cardiac heart valves, the skeletal muscle and the palate. During heart development, endocardial cells activated by factors secreted by the adjacent myocardium undergo EMT and invade through the basement membrane and into the cardiac jelly. The cells remain here as mesenchymal cells of the cardiac cushion and mediate development of the cardiac heart valves . In the context of the endocardium, TGF-β , BMP , β-catenin , Msx1/2  and Slug , among other factors, regulate endocardial EMT. EMT is also critical during myogenesis, particularly as the muscle precursor cells delaminate from the dermomyotome. The dermomyotome is an epithelial structure derived from the paraxial mesoderm. Pax3 expression is an important determinant of muscle cell progenitors in the dermomyotome , and interacts with a conserved gene network including Six, Eya and Dach proteins to direct muscle precursor development and migration . Upon induction of EMT, the muscle precursors invade into the developing limbs and differentiate into mature muscle cells. Interestingly, although the EMT associated with myogenesis has not been comprehensively studied, the genes identified as important in this EMT have not been implicated in other classical EMT processes such as gastrulation, neural crest formation, heart valve formation or palatogenesis. While the precise reason for this difference remains unknown, it suggests that the EMT associated with muscle development may be orchestrated in a slightly different manner than other developmental EMTs, potentially setting the stage for the muscle differentiation program. Future studies on the early events of myogenic precursor formation will investigate the nature of this EMT. Interestingly, during palatogenesis the function of EMT is also somewhat different than other developmental EMTs. Instead of giving rise to a migratory mesenchymal cell type while maintaining the epithelial source, during palatal fusion the epithelial tissue within the medial edge epithelium (MEE) collapses, giving rise to a continuous mesenchymal tissue and a seamless palate . Similar to other examples of developmental EMT, the molecular regulators of EMT in the palate include TGF-β , Snail  and Twist .
Interestingly, the reverse process, MET, is also observed during development, particularly in nephrogenesis as the ureteric bud invades the metanephric mesenchyme. Upon induction, the metanephric mesenchyme condenses around the invading bud and undergoes MET. The Six/Eya proteins are also implicated in the maintenance of the metanephric mesenchyme and the prevention of precocious differentiation and MET . Six2, which is downstream of Six1 in the developing kidney , is critical in the decision to differentiate or self-renew. Downregulation of Six2 is linked to MET, and its presence is believed to be required for the maintenance of early nephrogenic progenitor cells . Therefore EMT and MET are critical in the development of multiple organs, and utilize diverse and tissue specific molecular mechanisms.
Defining Epithelial Plasticity and Partial EMT in Development
During development, EMT provides the flexibility to convert between different cell phenotypes that are required for proper morphogenesis as discussed above. However, the development and morphogenesis of numerous tissues during embryogenesis requires epithelial plasticity that differs somewhat from classical EMT, and that parallels changes seen in cancer. However, similar to EMT, these processes require perturbations of the normally static nature of an epithelium and the acquisition of properties of invasive and motile cells. In particular, epithelial plasticity is essential during branching morphogenesis that occurs during development of numerous organs including the kidney, lung, mammary gland and salivary gland . Branching morphogenesis is a complex morphogenic mechanism by which a tubular epithelial structure bifurcates in an iterative process to generate an intricate ductal tree. During branching morphogenesis, tip cells located at the leading edge of the elongating epithelial tube undergo morphologic and cellular changes in response to extracellular signals, including FGFs, culminating in the bifurcation of the epithelial tube . While distinct from a complete EMT, during branching morphogenesis the tip epithelial cells transiently exhibit mesenchymal features, ranging from loss of apico-basal polarity, degradation of the basement membrane, and increased cell protrusions and mesenchymal marker expression, depending on the tissue . The plasticity of epithelial cells during development suggests that in the broadest sense, the difference between epithelial and mesenchymal cells may be more of a continuous spectrum rather than two independent cell states.
Epithelial Plasticity in the Mammary Gland
The mammary gland develops in three distinct stages consisting of an embryonic stage of rudimentary gland development, a pubertal stage of ductal elongation and branching, and a pregnancy stage of alveolar differentiation and tertiary ductal branching . Mammary gland development begins during mid-gestation as the mammary lines form and placodes develop along these lines. The placodes invaginate into the underlying subdermal stroma forming the mammary bud. As embryogenesis continues, the mammary bud begins to invade into the surrounding adipose tissue producing a rudimentary mammary gland that is arrested at this stage until puberty.
EMT in Breast Cancer
The prognosis of breast cancer patients is tightly correlated with the degree of spread beyond the primary tumor . However the mechanisms by which epithelial tumor cells escape from the primary tumor and colonize a distant site is not entirely understood. Recent work in breast cancer and other cancers has postulated EMT as a potential mechanism by which epithelial tumor cells acquire a more motile and invasive phenotype and escape from the primary tumor . In breast cancer, similar to other cancers, EMT rarely occurs homogenously across the whole tumor. The exceptions include diffuse lobular carcinoma, where E-cadherin is lost uniformly across the tumor and gene expression profiles identify genes involved in EMT including upregulation of fibronectin and collagen type I and III . Another exception is that of the sarcomatoid tumor of the breast, also referred to as spindle-cell carcinoma tumors, which display a clear mesenchymal and spindle cell phenotype  while still retaining keratin expression and evidence of an epithelial origin . However, based on the expression of EMT markers, recent evidence suggests that EMT also occurs in a more local manner at the invasive front of the tumor , and that such a localized EMT is no less clinically significant than the EMTs mentioned above. In this more limited example of EMT, the tumor cells are primed to undergo EMT by genetic and epigenetic changes. Then, with the proper extracellular inputs, including activation of TGF-β and Wnt signaling at the leading edge of the tumor coupled with the expression of EMT regulators such as Snail/Slug/Twist, Cripto-1 and Six1, the cells acquire a mesenchymal phenotype that permit them to locally invade and escape from the primary tumor, setting the stage for metastatic dissemination (Fig. 5b). However, once the epithelial tumor cells undergo EMT, they may be phenotypically indistinguishable from fibroblasts. As a consequence, unequivocal evidence of EMT in most cancers is lacking. The absence of pathologic evidence for EMT at the secondary site, since most metastatic lesions exhibit an epithelial phenotype, has also contributed to the disbelief by some that EMT occurs in tumor progression. Evidence for the role of EMT in cancer is complicated by the fact that at the secondary site the metastatic cells likely undergo a reversion or MET, permitting colonization of the distant site. Therefore in the course of cancer progression, EMT is a transient and reversible process. However, the recent use of cell fate mapping of epithelial tumor cells in the Whey Acidic Protein (WAP)-Myc transgenic mice provides direct evidence of EMT in the local invasion of tumor cells in breast cancer , finally putting an end to the argument as to whether EMT can occur in mammary/breast cancer. Furthermore, recent evidence obtained from microarray analysis of breast tumors support the hypothesis that certain types of breast cancer cells are primed to undergo EMT and spread. For instance, the basal or triple negative breast cancer subtype exhibits an aggressive phenotype and also correlates with the poorest clinical outcomes . Markers of EMT are preferentially displayed in the basal subtype as well as in a newly recognized claudin-low subtype, linking EMT with the molecular subtype of a tumor [74, 75]. When breast cancer cells lines are similarly classified, those cells classified as basal B, a subdivision of the basal cluster, also display a mesenchymal phenotype . This work supports the existence of EMT in breast cancer and suggests that the cells undergo MET at the site of metastatic colonization. This hypothesis underscores the necessary plasticity of cancer cells and suggests that instead of a permanent mesenchymal phenotype, migrating cancer cells likely exhibit epithelial plasticity that is reversible and dependent on the local microenvironment.
Recently, a novel hypothesis for the role of EMT in cancer progression has been proposed, whereby epithelial and mesenchymal cells cooperate during metastatic dissemination, potentially eliminating the need for MET at the distant site . According to this hypothesis, neither epithelial nor mesenchymal cells alone are sufficient to induce metastatic spread, however, when combined these cells cooperate to induce metastases. Evidence supporting this theory is derived from a model where EMT was induced through expression of p12CDK2-AP1 in a keratinocyte cell line. Neither control cells nor cells induced to undergo EMT by p12 expression alone were competent to develop metastases after subcutaneous injection in a xenograft model of squamous cell carcinoma. However, tumors derived from cells that had undergone EMT did exhibit local invasion and evidence of intravasation into the blood stream. When the same control cells and cells induced to undergo EMT were tested in an experimental metastasis model and injected directly into the vein of mice, only the control cells that did not undergo EMT could form lung metastases. However unexpectedly, when a 1:1 mixture of control cells and cells that had undergone EMT were injected subcutaneously, lung metastases developed that were composed solely of the control cells . These observations suggest that EMT is necessary for metastasis, but that it is not sufficient, and that it is the non-EMT cells that colonize distant tissues. Similarly, the findings suggest that non-EMT cells are necessary, but not sufficient for metastasis, and require cells that have undergone EMT in order to gain access to the circulation from the primary tumor . To provide conclusive evidence in support of this finding, however, will require fate mapping the EMT and non-EMT cells and following their phenotypic profile during the course of metastatic spread. However, these results suggest that cooperativity between cells of different phenotypes either through direct interaction or a secreted signal may be an important mechanism of metastatic spread.
In addition to the induction of EMT, which results in single cell dissemination of tumor cells, collective migration of groups of tumor cells may also be a mechanism by which cancer can spread . Clinical evidence has identified strands and columns of tumor cells in the peritumoral tissue that appears to be migrating away from the primary tumor as a collective group . In addition, growth of explanted tumors in three dimensional collagen gels identified groups of cells detaching from the primary tumor and invading into the surrounding matrix . Most recently, intra-vital, non-invasive imaging of tumor cell migration and invasion has supported the role of collective cell migration of tumor cells in vivo in a fibrosarcoma tumor model which is also consistent with observations of collective cell migration observed in a breast cancer xenograft mouse model [81, 82]. Analogous to the collective migration seen in mammary gland development and wound healing, the combination of epithelial plasticity while still maintaining adherence to neighboring cells appears to be another mechanism of tumor spread. Interestingly, the dominant mode of migration of a tumor may determine the preferred site of metastatic spread. Whereas solitary movement of tumor cells in vivo, particularly in cooperation with macrophages, directs tumor cells to the blood vessels resulting in hematogenous dissemination [83, 84], collectively migrating cells have been reported to result in lymphatic dissemination . Interestingly, TGF-β signaling, which induces EMT and single cell migration, is necessary for intravasation, extravasation and blood borne metastasis [82, 85]. In line with this finding, inhibition of TGF-β signaling in a rat mammary carcinoma model can inhibit single cell migration, but collective migration of tumor cells is maintained and results in exclusively lymphatic metastases . Together, these results suggest that the mode of migration may determine the route of metastatic dissemination.
Finally, the induction of EMT not only regulates features intrinsic to the tumor cells, but also regulates the tumor cell’s interaction with its microenvironment, including the immune cells. Tumor cells that have undergone EMT in response to forced expression of Snail induce immunosuppressive cytokines and impair tumor surveillance mechanisms . Again, the ability of cells undergoing EMT to evade the immune system by directly regulating it, more than likely contributes to the aggressiveness of the tumor. Overall, these results suggest that the induction of EMT is not simply a migratory mechanism of local spread of tumor cells from the primary site, but instead induces a comprehensive program of properties necessary for tumor progression. Additionally, these results reinforce the viability of therapeutic agents that target regulators of EMT/tumor-initiating cells, particularly those developmental EMT regulators expressed de novo in cancer.
Molecular Pathways of Development Reactivated in Breast Cancer
Recent work has identified numerous developmental regulators of EMT that are misexpressed in breast cancer and contribute to breast cancer progression. The spectrum of EMT inducers implicated in breast cancer includes both signaling pathways such as TGF-β, Wnt/β-catenin, and Cripto, as well as transcription factors including Twist, Snail/Slug, Lbx1, and Six1. In addition, recently non-coding RNAs, including microRNAs, have been implicated in EMT in cancer and development [93, 94]. For a more detailed review of microRNAs in EMT, please see the accompanying review by Goodall and colleagues.
During mammary gland development, numerous factors have been implicated in branching morphogenesis. Interestingly, a number of these factors have also been implicated in oncogenic EMT during breast cancer progression, including Cripto-1. Cripto, a member of the epidermal growth factor-Cripto/FRL-1/Cryptic (EGF-CFC) family of proteins, is a developmental regulator during early embryogenesis and a cofactor for the TGF-β family ligand, Nodal. Cripto is expressed at the primitive streak and Cripto-knockout mice do not progress beyond early body patterning events due to a deficiency in formation of the mesoderm suggesting a role for Cripto in the early developmental cell movements and EMT . However, Cripto is also expressed in the epiblast epithelium prior to gastrulation suggesting the Cripto likely cooperates with other pathways and is not sufficient alone to induce EMT . In addition, Cripto is a developmental regulator of branching morphogenesis in the mammary gland. Cripto-1 expression is observed in the cap cells in the terminal end buds of the mammary gland , and Mouse Mammary Tumor Virus (MMTV)-Cripto transgenic mice display increased mammary gland branching . These data suggest that Cripto is expressed in the cells displaying epithelial plasticity during ductal branching and that its misexpression increases branching. Additional work in vitro confirms that Cripto overexpression in numerous mammary epithelial cell lines can increase branching in three-dimensional cultures, increase the invasive and migratory properties of the cells, and induce an EMT . Not only can Cripto induce these properties of EMT in vitro, but its misexpression is also sufficient to induce mammary tumor formation in transgenic mice. Interestingly, MMTV-Cripto transgenic mice develop tumors that display properties of EMT including downregulation of E-cadherin and upregulation of vimentin and N-cadherin . Importantly, transgenic mice expressing Cripto under the regulation of the WAP promoter also develop mammary tumors and the tumors that arise are of multiple histological subtypes including glandular, papillary, adenosquamous, solid and myoepithelioma . The mixture of histologic subtypes suggests that Cripto expression may not only induce an EMT, but it may also induce a stem/progenitor-like phenotype that would be consistent with other regulators of EMT . In support of a role for Cripto misexpression in human breast cancer, increased Cripto expression has been observed in 50% of ductal carcinoma in situ and an even greater 80% of infiltrating ductal carcinoma . Paralleling the observations from early embryonic development, the expression of Cripto in breast cancer prior to the acquisition of invasive properties suggests that Cripto expression likely cooperates with other pathways to induce an invasive phenotype and may prime the cells for EMT. Together, these results establish Cripto as a significant regulator of branching morphogenesis in the mammary gland and demonstrate that misexpression of Cripto in breast cancer models induces EMT. Importantly, Cripto misexpression correlates with advanced disease in human cancer, reinforcing the significance of its aberrant expression in breast cancer progression.
Recent work has begun to elucidate the potential mechanism of Cripto misexpression and, not surprisingly, another EMT regulator and mammary developmental gene, Msx2, upregulates Cripto . Under the regulation of progesterone, the homeodomain protein Msx2 increases lateral branching and transgenic mice expressing Msx2 in the mammary gland also display increased branching similar to what is observed with misexpression of Cripto . In the context of cancer, Msx2 induces EMT in vitro in numerous cancer cell lines [64, 102]. Finally, analysis of clinical human breast cancer samples revealed that Msx2 expression is specifically observed in the infiltrating cells of the tumor that display an invasive phenotype . Together, these data suggest that the developmental gene network consisting of Msx2 and Cripto is activated and likely contributes to mammary tumor progression. For a more detailed discussion of Cripto and the role of EGF signaling in EMT and breast cancer, please see the accompanying review by Salomon and colleagues.
The E-cadherin Repressors Snail, Slug and Twist
The E-cadherin repressors, Snail and Slug, and the basic helix-loop-helix transcription factor Twist, are also clear examples of inappropriately expressed developmental regulators in breast cancer. In the primitive streak, Snail is expressed in response to FGF, resulting in the transcriptional downregulation of E-cadherin and the induction of EMT during gastrulation . In mice lacking Snail, the cells of the primitive streak begin to invade, however the EMT, specifically the downregulation of E-cadherin, is incomplete. The remaining cells maintain an epithelial phenotype including apico-basal polarity and cell-cell adhesions . In addition to gastrulation, the E-cadherin repressor proteins are also expressed during other developmental EMT events including palatal fusion (Snail and Twist) and neural crest migration (Snail and Slug depending on the species) . Most recently, microarray analysis of microdissected terminal end buds in the mammary gland identified a significant upregulation of the Snail and Twist transcripts when compared to their expression in mature ducts, suggesting that these developmental EMT regulators may also regulate epithelial plasticity in the mammary gland . Snail, Slug and Twist are also significant inducers of EMT in cancer cells and repress E-cadherin expression [35, 104, 105, 106]. However, all three transcription factors also regulate other aspects of EMT. For example, Snail and Slug regulate tight junction stability , gap junctional protein expression , desmosome disassembly  and protease expression . Indeed, Snail and Slug are expressed in response to numerous EMT inducing stimuli including Wnt signaling , Lbx1 , TGF-β signaling , loss of Sim2 expression , and hypoxia . Similarly, Twist not only represses E-cadherin, but also induces mesenchymal gene expression and initiates invasion . Like Snail and Slug, Twist is the target of numerous EMT-inducing stimuli including hypoxia and Msx2 [102, 114]. The convergence of numerous EMT pathways on Snail, Slug and Twist suggest that they are critical nodes in the network of EMT signaling.
Importantly, the association of Snail, Slug, and Twist with EMT in vitro is also consistent with their observed correlation with poor prognosis in human breast cancers. In infiltrating ductal carcinoma, Snail expression correlates with lymph node metastasis and with decreased relapse free survival [115, 116], while Slug correlates with metastasis . Again, like Snail and Slug, Twist is associated with adverse outcomes in breast cancer: it correlates with invasive lobular carcinoma and decreased disease specific survival [35, 117]. Overall, Twist, Snail and Slug all can induce EMT in the context of normal tissue and cancer. Importantly, in cancer the expression of these proteins correlates with aggressive disease, reinforcing the pro-metastatic effects of an oncogenic EMT.
The Six Family of Homeodomain Proteins
The E-cadherin repressors are not the only transcription factors that play an important role during developmental EMT and that are inappropriately expressed in breast cancer. The Six family of homeodomain transcription factors, specifically Six1, Six2 and Six4, are also important during developmental EMT. For example, in muscle development, the homeodomain transcription factors Six1 and Six4 regulate migration and cell fate determination of muscle precursor cells [40, 118]. Specifically, the Six1/Six4 double knockout mouse exhibits a defect in the delamination and migration of muscle precursor cells, resulting in the absence or hypoplasia of muscles  and underscoring the importance of Six1 and Six4 in the EMT that occurs during myogenesis. In kidney development, Six1 and Six2 are necessary for kidney formation [57, 58]. In particular, Six2 maintains the mesenchymal phenotype of renal progenitor cells and the downregulation of Six2 is necessary for the MET observed as metanephric mesenchymal cells transition into epithelial cells destined to form the tubule epithelium. These data demonstrate that Six2 is an important regulator of the mesenchymal state. Interestingly, not only is Six2 critical in maintaining the mesenchymal phenotype, but it is also necessary for maintaining the nephrogenic progenitor cell population in the kidney, reinforcing the link between a mesenchymal and a progenitor/stem cell phenotype . Importantly for this review, the Six1 transcription factor is dynamically expressed in the embryonic and postnatal mammary gland, exhibiting its highest expression in the embryonic, neonatal, and pubertal mammary glands, after which its expression dramatically diminishes. Surprisingly, knockout of Six1 in the mammary gland does not appear to have a significant functional consequence on mammary gland development. However, due to the compensatory increase in Six2 and Six4 expression in response to Six1 loss, and thus the likelihood of functional redundancy, the function of the Six family in mammary gland development remains unclear . In addition to the role of the Six family members in epithelial plasticity during muscle and kidney development, Six1 induces features of EMT in both a mammary carcinoma cell line and in mammary tumors derived from mammary specific Six1 overexpressing transgenic mice [36, 37]. Consistent with other regulators of EMT, Six1 also induces metastatic spread in mouse models of tumor progression, and analogous to the function of Six2 in development, Six1 overexpression induces properties of mammary and mammary cancer stem/progenitor cells [36, 37]. The underlying mechanism of Six1-induced EMT and metastasis appears to be, at least in part, through the activation of TGF-β signaling , a pathway that is well -established as a strong inducer of EMT in breast cancer . As seen with Snail, Slug, and Twist, increased Six1 expression also correlates with poor prognosis in breast cancer patients . Patients with elevated levels of Six1 have increased lymph node involvement, shortened time to relapse and metastasis, and decreased survival . Together this work demonstrates that misexpression of the Six1 developmental EMT regulator in breast cancer induces an analogous EMT program in tumor cells that contributes to tumor progression.
The Homeodomain Protein Lbx1
Although genes that play a role in mammary gland development are misexpressed in breast cancer, so are developmental regulators of EMT that are not known to be involved in mammary gland development. Similar to the Six family members, Lbx1 is a well established regulator of muscle precursor migration and cell fate during development [11, 120]. Recent work has shown that Lbx1 expression in mammary carcinoma cells induces properties of EMT and progenitor cells, activates known inducers of EMT, and correlates with the basal subtype of human breast cancer . Together these results suggest that EMT programs beyond those critical for mammary gland development may be activated in human breast cancer and contribute to an oncogenic EMT and breast cancer aggressiveness.
Sim2 Transcription Factor
In contrast to the inducers of EMT, inhibitors of epithelial plasticity may be downregulated during development and cancer, leading to an induction of a more mesenchymal phenotype. Sim2 is a protein involved in mammary gland development and breast cancer progression, likely through its regulation of epithelial plasticity. Sim2 is a basic helix loop helix/Per-Amt-Sim (bHLH/PAS) transcription factor which has been identified as an important developmental transcriptional repressor . In the mammary gland, loss of expression of the short isoform of Sim2 (Sim2s) disrupts both duct hollowing and morphogenesis, induces the breakdown of the basement membrane, leading to the loss of E-cadherin and apico-basal polarity, and results in the invasion of epithelial cells into the surrounding stroma. These changes are all consistent with Sim2 repressing branching through the maintenance of an epithelial phenotype . In the context of cancer, loss of Sim2 in mammary carcinoma cells also induces an EMT as measured by the loss of E-cadherin and concomitant gain of vimentin and N-cadherin . Importantly, Sim2 is lost in the majority of breast cancers , suggesting that in the absence of Sim2 breast cancer cells may be primed for epithelial plasticity and EMT. Therefore, Sim2 is example of a developmental mammary gland regulator of epithelial plasticity that when lost in cancer induces features of EMT which likely contribute to metastatic spread.
Developmental EMT Signaling Pathways Reactivated in Breast Cancer
In addition to transcription factors, cell signaling pathways are also critical inducers of EMT in the context of development and in cancer. One of the best studied EMT signaling pathways is TGF-β signaling. TGF-β is a ubiquitously expressed cytokine that binds to a target cell through the type I and II TGF-β receptors, initiating multiple signaling cascades, including the canonical Smad signaling pathway, that ultimately regulate transcription in combination with cofactors . During development, TGF-β is required for heart valve formation, where TGF-β2 activates the Type I receptor to induce an EMT and permit the invasion of endocardial cells into the cardiac cushions [124, 125]. Not only does TGF-β induce EMT in heart development, but it also induces EMT during palatogenesis, where TGF-β3 is required for palatal fusion [8, 126]. In vitro, TGF-β has been established as a classic inducer of EMT in a wide range of systems . The consequences of TGF-β treatment are dramatic, leading to a loss of apico-basal polarity , downregulation of cell-cell adhesions , expression of mesenchymal cytoskeletal proteins including vimentin , as well as extracellular proteases . In breast cancer, TGF-β signaling plays a dual role in tumorigenesis. In early lesions, TGF-β is a significant tumor suppressor through its growth inhibitory effects . In contrast, TGF-β promotes metastatic spread in later stages of tumorigenesis, likely at least in part through its ability to induce EMT [85, 130, 131]. In numerous models of breast cancer associated invasion and metastasis, activated TGF-β signaling induces increased aggressiveness. For example, in mice overexpressing the Neu oncogene, activated TGF-β signaling increases the number of lung metastases even while decreasing the growth of the primary tumor . Likewise, ablation of TGF-β signaling in the same model decreases lung metastasis while also decreasing the latency of primary tumor growth, again emphasizing the dual functions of TGF-β signaling in tumorigenesis . Additionally, clinical evidence suggests a correlation between expression of the TGF-β ligands and poor patient outcome [132, 133]. Furthermore, activated TGF-β signaling has been observed in breast cancer bone metastases and contributes to the establishment of these lesions . Because TGF-β signaling is critical for EMT during cardiogenesis and palatogenesis, and because it also promotes EMT in a tumorigenic context, it is likely that the ability of TGF-β to promote metastasis is at least in part due to of its ability to induce an EMT. For a more detailed description of the role of TGF-β signaling in EMT, please see the accompanying review by Schiemann and colleagues.
Similar to the TGF-β pathway, the Wnt/β-catenin pathway is also implicated in EMT and epithelial plasticity during development and cancer. The Wnt pathway is activated during multiple stages of mammary development from the earliest stage during embryogenesis where Wnt signaling is necessary for mammary bud formation, to the alveolar differentiation and side branching observed during pregnancy. Wnt signaling likely plays a role in numerous aspects of mammary development including cell fate determination, maintenance of mammary progenitor cell populations, branching morphogenesis and alveolar differentiation . Cells composing the TEB express multiple Wnt ligands and receptors, consistent with a role for the Wnt pathway in normal TEB function . Indeed, misexpression of Wnt-1 under the control of the MMTV promoter induces hyperbranching and precocious alveolar differentiation . Not only is the Wnt pathway important for normal mammary development, but it also regulates EMT in other developmental contexts including gastrulation and cardiac valve formation [51, 137, 138]. Furthermore, in vitro studies confirm that the Wnt/β-catenin pathway is capable of inducing epithelial plasticity. Activation of the Wnt pathway in breast cancer cells induces the EMT regulator, Snail, and upregulates the mesenchymal marker, vimentin [109, 139]. Additionally, the Wnt/β-catenin pathway induces EMT in numerous models including mammary epithelial and carcinoma cell lines [32, 109]. Importantly, markers indicating active Wnt/β-catenin signaling, including nuclear β-catenin, also correlate clinically in breast cancer patients with poor prognosis [41, 42]. Taken together, these data implicate the Wnt/β-catenin pathway in epithelial plasticity and EMT in both development and cancer.
The expression of these numerous developmental EMT regulators in breast cancer, coupled with the induction of a motile and mesenchymal phenotype in breast cancer cells in response to the activation of these developmental genes and pathways, establishes a significant parallel between development and cancer. Most importantly, the significance of this misexpression is revealed by the clear clinical implications of the inappropriate activation of these genes and pathways. In sum, the concept of an oncogenic EMT and its resemblance to developmental EMT is an important framework for understanding the mechanisms of cancer metastasis and will likely provide new therapeutic targets.
The spread of cancer cells from the primary tumor is a major complicating factor of cancer progression, preventing effective treatment and substantially decreasing patient prognosis. Only recently have we begun to understand the mechanism of tumor cell spread and to identify potential therapeutic targets to prevent or limit the extent of metastatic dissemination. As the mechanisms of spread in epithelial-derived cancer are becoming clear, similarities have been recognized to developmental programs of migration and invasion. These similarities have led to the hypothesis that in tumorigenesis, developmental pathways are activated inappropriately that contribute to tumor progression. EMT is a critical mechanism of migration and invasion during development. Recently an analogous process has been observed in cancer cells employing embryonic pathways as a mechanism to enhance motility and invasion. Importantly, features of oncogenic EMT correlate with clinical parameters, suggesting that reactivation of the developmental EMT program in cancer contributes to cancer progression. The reactivation of embryonic programs that are potentially not functional in normal adult tissue provides an opportunity to selectively target these pathways in cancer cells while conferring minimal side effects. Future work will continue to investigate the parallels between development and cancer and begin to consider oncogenic EMT pathways as potential therapeutic targets.
Acknowledgement of Financial Support: D.S.M. was funded by a predoctoral fellowship from the Department of Defense Breast Cancer Research Program (W81XWH-06-1-0757). SMF is funded by a predoctoral fellowship from the Department of Defense Breast Cancer Research Program (W81XWH-08-1-0332). HLF is funded by grants from the National Cancer Institute (2RO1-CA095277), The American Cancer Society (#RSG-07-183-01-DDC), and The Susan G. Komen Foundation (BCTR0707562).
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