A major challenge in halting carcinoma progression comes from the intrinsic heterogeneity of the tumors. These are composed of distinct cell types, including differentiated cancer cells and cancer stem cells (CSCs), as well as cancer-associated fibroblasts (CAFs), leukocytes, and endothelial cells. Noncancer stromal cells and interstitial extracellular matrix (ECM) in the tumor microenvironment sustain and instruct tumor cells by providing mechanical support, communicating through cell contacts, and secreting molecules, such as growth factors . Understanding the complex relationships and signaling networks between the different cells in a tumor is essential to target cancer cells with adequate therapies. While cancer cells are marked by genetic and epigenetic alterations, tumor progression largely depends on the signaling networks that direct cell viability, and tumor growth and dissemination. Cytokines in the tumor microenvironment protect cancer cells against the immune system, and growth factors such as those acting through tyrosine kinase receptors promote tumor growth. In addition, new blood vessel formation in the tumor correlates with tumor survival and growth by facilitating uptake of glucose, nutrients, and oxygen [1,2].
Tumor cell dissemination is critical in cancer progression and involves multiple processes, leading to the generation of metastases at remote loci [3,4]. Cancer dissemination is initiated by decreased cell–cell adhesion, increased motility and invasive properties that allow carcinoma cells to detach from the primary tumor and invade surrounding tissue, through collective or individual cell migration . The individualization, migration, and invasive properties of epithelial cancer cells result from epithelial–mesenchymal transition (EMT), a transdifferentiation process that is integral to normal development, yet can be reactivated by carcinoma cells to aid cancer progression . Conversely, cancer cells that have acquired mesenchymal properties can revert to an epithelial state through mesenchymal–epithelial transition (MET), after reaching a secondary colonization site, thus reacquiring the properties of the original tumor following metastasis.
TGF-β has emerged as a potent secreted factor that drives cancer progression, not only through its immunosuppressive and proangiogenic roles, but also perhaps more importantly as a potent inducer of epithelial plasticity leading to EMT [7,8]. These activities complement the ability of TGF-β to act as tumor suppressor through its ability to inhibit epithelial cell proliferation. However, genetic mutations in TGF-β signaling components or changes in signaling pathways often inactivate TGF-β's growth inhibitory activities, thus making room for TGF-β to aid in cancer progression. Increased levels of TGF-β expression or activation, or increased TGF-β receptor levels in the cancer cells, result in increased autocrine TGF-β signaling in the tumor microenvironment, and enable cancer cells to use TGF-β signaling as a key to survive and escape the primary tumor to form metastasis [8,9].
TGF-β SIGNALING AND EPITHELIAL–MESENCHYMAL TRANSITION
TGF-β signaling is initiated upon interaction of TGF-β with a tetrameric cell surface complex of two type II and two type I transmembrane kinase receptors, TβRII and TβRI. Following ligand binding, the TβRII receptors activate TβRI receptors through direct phosphorylation. These in turn phosphorylate the intracellular mediators Smad2 and Smad3 proteins at two C-terminal serines. Two activated Smad proteins then combine with one Smad4 to form trimeric Smad complexes that translocate into the nucleus and regulate target gene expression. Smads activate or repress transcription through physical interactions with high-affinity DNA-binding transcription factors, and coactivators and corepressors, at regulatory gene sequences [10,11]. In addition to Smad signaling, TGF-β also initiates non-Smad signaling from the TβRII/TβRI complexes, leading to the activation of pathways that are more commonly seen as effectors of receptor tyrosine kinase signaling, such as PI3K/Akt, Erk, and p38 MAPK, and Rho-GTPases pathways [12,13].
Cancer cell invasion leading to tumor cell dissemination is facilitated by an epithelial plasticity response leading to EMT. Studies of EMT in cancer cells and in normal development, as well as in cell culture, illustrate that EMT results from a complex series of cellular and molecular changes that are regulated at the transcription, posttranscriptional, and posttranslational levels [6,14] (Fig. 1). Depending on the epithelial cell type and tissue context, EMT may come with variations of underlying mechanisms, and the extent to which epithelial cells transition into a mesenchymal state may differ as well. Yet, EMT results from a clear complement of parallel and consecutive changes in signaling, gene expression, and cell phenotype and behavior that highlight a common EMT program. EMT starts with dissolution of epithelial cell–cell contacts and loss of apical–basal polarity  (Fig. 1a). In addition, the expression of epithelial junction proteins and epithelial markers is downregulated at the level of gene expression, concomitantly with increases in the expression of mesenchymal adhesion and marker proteins . Three families of transcription factors potently regulate this epithelial to mesenchymal switch in gene expression, that is, the Snail/Slug, ZEB1/2, and Twist families, although several other transcription factors have also been shown to direct or contribute to EMT programs . In addition, the actin cytoskeleton is dramatically reorganized and phenotypic changes occur to enable the cell to acquire a front-rear polarity and motility. Finally, the cells acquire invasive properties through increased expression and secretion of metalloproteases (MMPs), and cells that underwent EMT are also efficiently capable of synthesizing components of the ECM to reconstitute their microenvironment following cell invasion [15,18,19].
ACTIVATION OF EPITHELIAL–MESENCHYMAL TRANSITION IN RESPONSE TO TGF-β SIGNALING
TGF-β acts as a common and potent inducer of EMT by combining both Smad and non-Smad signaling pathways [7,20,21] (Fig. 1b). TGF-β signaling directly activates the expression of the EMT transcription factors, Snail and Slug, ZEB1 and ZEB2, and Twist [7,17,21]. For example, the Smad3/Smad4 complex binds directly to regulatory promoter sequences of Snail, inducing its transcription, and, subsequently, an active complex formed by Smad3/Smad4 and Snail can bind to regulatory promoter sequences of the genes encoding the epithelial junction proteins E-cadherin and occludin, leading to TGF-β-induced repression of their expression . Smad signaling also leads to the expression of ZEB transcription factors [7,17,21], which in turn repress the expression of the miR-200 family, a microRNA family that, in a feedback loop, also targets and inhibits the expression of ZEB proteins, as well as TβRI and Smad2, at the mRNA level. As a result, the ZEB1 and ZEB2 mRNA and protein levels are further increased and promote EMT responses . In addition, TGF-β regulates the expression of MMPs such as MMP2 and MMP9, as well as components of the ECM such as fibronectin and collagens, most likely through activities of EMT transcription factors [7,21].
Activation of EMT transcription factor expression in response to TGF-β has also been linked to alternative splicing in EMT . Indeed, ZEB1 and ZEB2, whose expression is activated by TGF-β, repress the expression of the RNA-binding proteins ESRP1 and ESRP2 [25▪]. Their downregulation during EMT leads to broad alterations of splicing patterns that involve cell–cell adhesion or actin cytoskeleton proteins, and characterize the mesenchymal state. Downregulation of ESRP1 and ESRP2 also leads to a switch in FGF receptor isoform expression toward a mesenchymal form of this receptor, which enhances EMT progression and cell invasion [26▪].
The expression of EMT transcription factors and their targets is additionally regulated by epigenetic changes . For example, the lysine-specific histone demethylase LSD1 is recruited by Snail at the E-cadherin promoter and mediates a decrease in H3K4m2 levels that partially inhibits promoter activity and contributes to downregulation of E-cadherin expression . Additionally, the histone methyltransferase MMSET was shown to bind to the TWIST1 locus, and MMSET overexpression in prostate epithelial cells correlates with increased H3K36me2 levels, Twist1 expression, and EMT [28▪]. Both LSD1 and MMSET are expressed in solid tumors such as breast tumors at higher levels than in normal epithelial cells [29,30]. Furthermore, H3K9me2 methylation of the E-cadherin gene is mediated by cooperation of Snail with the methyltransferase G9a and facilitates reversible DNA methylation at regulatory promoter sequences in TGF-β-induced EMT in immortalized mammary epithelial cells [31▪]. These reports suggest that inhibitors against these chromatin modifiers may represent novel therapeutic opportunities against the progression of cancers. In how far epigenetic regulation of EMT transcription factors is mechanistically linked to activation of TGF-β/Smad signaling remains to be determined.
In addition to the regulation of miR-200 expression by ZEB1 or ZEB2, and its feedback on ZEB1/2 expression, in response to TGF-β, the EMT differentiation program is further controlled by a network of microRNAs that helps define changes in cytoskeleton reorganization, epithelial polarity, and intracellular signaling. For example, miR-155 expression is directly activated in response to TGF-β through Smad signaling and targets RhoA to facilitate tight junction dissolution during EMT. Accordingly, downregulation of miR-155 expression inhibits TGF-β-induced EMT . TGF-β also induces miR-491-5p expression, which targets the polarity protein Par3 and disrupts tight junctions . Other microRNAs directly target components of the TGF-β signaling pathway. Thus, miR-302, miR-372, and miR-204 directly repress TβRII expression [34▪,35]. Finally, other microRNAs act as tumor suppressors through inhibition of EMT and may therefore serve as therapeutic tools against cancer progression .
TGF-β-induced non-Smad signaling also promotes the EMT program [7,20,21] (Fig. 1b). TGF-β activates the PI3K/Akt/mTOR pathway, which, through the activity of mTOR complex 1, results in increased protein synthesis, motility, and cell invasion during EMT . Activation of mTOR complex 2 and regulation of Rho-like GTPases by TGF-β are necessary to proceed with the cytoskeletal reorganization, and thus allow transition of the cells toward the mesenchymal phenotype. Consequently, inhibition of mTOR complex 2 activity dramatically decreases cancer cell dissemination, likely through inhibition of TGF-β-induced EMT [38▪]. Akt activation in response to TGF-β also results in phosphorylation of the ribonucleoprotein hnRNPE1, a component of a complex that interacts with the 3′ untranslated regions of select mRNAs and thus inhibits translation. hnRNPE1 phosphorylation then results in dissociation of the complex from the mRNAs encoding Dab2 and ILEI, thus reversing translation inhibition, and allowing Dab2 and ILEI to contribute to EMT and metastasis [39▪,40▪].
Finally, posttranslational modifications have been shown to regulate the stability of EMT transcription factors, and consequently EMT progression. F-box family proteins act as E3 ubiquitin ligases that target Snail, ZEB, and Twist, leading to their ubiquitylation and proteosomal degradation, which may be seen as a mechanism to terminate EMT and set the stage for reversal to the epithelial phenotype [41,42]. Additionally, TGF-β directly activates, by binding of the Smad3/4 complex to regulatory promoter sequences, the expression of the E3 ubiquitin ligase HDM2, resulting in ubiquitylation and degradation of p53, which correlates with EMT progression . In addition to ubiquitylation, sumoylation also regulates TGF-β-induced EMT. During EMT, TGF-β signaling leads to decreased expression of the SUMO E3 ligase PIAS1. As a result, the level of sumoylated SnoN, which antagonizes TGF-β-mediated EMT responses, is decreased, enhancing EMT progression .
Importantly, other growth factors such as EGF or FGF, which act through receptor tyrosine kinases and are released into the tumor microenvironment, can also synergize with TGF-β signaling to enhance EMT responses [7,21]. Therefore, the relative abundance and contributions of different signaling molecules present in the tumor microenvironment may serve as critical determinants of cancer progression by triggering EMT and tumor cell dissemination.
TGF-β-INDUCED EPITHELIAL–MESENCHYMAL TRANSITION IN CANCER INVASION AND DISSEMINATION
In carcinoma progression, EMT is believed to benefit tumor behavior and dissemination at several levels [3,6,14,45] (Fig. 2). It plays a key role in initiating carcinoma cell invasion that will lead to cancer cell dissemination [6,14,45]. EMT also confers resistance to cell death and has been linked to resistance to chemotherapy or escape from immune surveillance . Additionally, TGF-β-induced EMT correlates with acquisition of CSC-like properties [46,47,48▪▪]. Finally, EMT plays an important role in the generation of stromal cells that support and instruct cancer progression .
The development of novel imaging techniques has illuminated the processes of tumor cell movement which leads to metastasis [49,50]. Local invasion of cancer cells is critical to the initiation of the multistep process that enables cancer cells to metastasize . In the process of invasion, cancer cells move either individually or collectively [51,52]. EMT drives local invasion and metastasis, and is a prerequisite for individual cell invasion. In collective cell invasion, the migrating and invading cells stay together, and maintain epithelial marker expression, cell–cell junctions, and cell–matrix adhesion, yet the leading cells at the edges show an epithelial plasticity response resulting in loss of epithelial integrity . Collective cell invasion characterizes cancers that are rarely metastatic, whereas individual cell invasion allows for the dissemination of cancer cells to distant sites of metastasis . Initiating the process that leads to tumor metastasis, carcinoma cells activate the EMT program to achieve initial invasion and dissemination. Once the mesenchymal cells have settled at the sites of metastasis, they may revert to an epithelial phenotype to form tumor colonies with epithelial characteristics that are similar to the original primary tumor . Induction of EMT in response to increased TGF-β activity and signaling is thought to provide important functions in the initiation of metastasis in this model.
Using intravital imaging, TGF-β signaling was shown to be a key determinant in cancer cell behavior, that is, in defining whether cancer cells will migrate collectively or individually. TGF-β signaling is transiently and locally activated in motile single breast carcinoma cells, and this activation results in a switch from collective to individual cell migration and to promoting distant metastasis [54▪▪]. This TGF-β-induced switch requires activation of a Smad-mediated transcription program, yet other factors, for example, ligands that induce EGF receptor activation, contribute to the increased motility of cancer cells. In the model system used for this study, forced and prolonged activation of TGF-β signaling promotes single-cell invasion in vivo, yet does not promote lung metastasis. Instead, TGF-β signaling needs to be reduced for later stages of the metastatic process [54▪▪]. These findings highlight the importance of transient and reversible EMT induced by TGF-β in invasion and metastasis.
Several reports revealed the mechanisms of initiation of invasion by TGF-β-induced EMT that are dependent on cytoskeletal changes. Formation of invadopodia, which are actin polymerization-dependent protrusions, is an aspect of the EMT process that is essential for tumor cell invasion and intravasation [55▪,56,57]. In TGF-β-induced EMT of MCF10A breast epithelial cells, the focal adhesion protein Hic-5 mediates invadopodia formation, which leads to increased ECM degradation, and cell motility and invasion [58▪]. TGF-β-induced EMT additionally results in an isoform switch from myosin IIC to myosin IIB expression and phosphorylation of myosin heavy chain IIA [59▪]. These changes in expression and phosphorylation of myosin are critical for increased cell invasion.
EPITHELIAL–MESENCHYMAL TRANSITION AND CANCER STEM CELLS
CSCs, a small population of self-renewing cells with the ability to initiate tumor formation, are increasingly seen as having a key role in driving cancer progression, as they give rise to heterogeneous cancer cell populations [60,61]. In contrast to a hierarchical, unidirectional model of CSC differentiation, the inherent differentiation plasticity of carcinoma cells allows differentiated cancer cells to acquire CSC-like properties and vice versa . Several reports demonstrate that TGF-β-induced EMT induces carcinoma cell dedifferentiation and leads to the generation of CSCs from differentiated cancer cells. In human breast cancers, a small subpopulation of cancer cells with a CD44high/CD24low antigenic phenotype was shown to exhibit CSC properties . Stem-cell-like cells, isolated based on this phenotype, from mouse or human mammary carcinomas were shown to express EMT markers . Transformed human mammary epithelial cells that have undergone EMT show an increased ability to form mammospheres, soft agar colonies, and tumors, which correlates with their CSC properties [46,47]. Furthermore, a spontaneously arising mesenchymal subpopulation of immortalized human mammary epithelial cells requires autocrine TGF-β signaling and Wnt signaling for the maintenance of its mesenchymal state and tumorigenicity, thus linking TGF-β with EMT and CSCs. In this cell system, bone morphogenetic protein (BMP) antagonizes TGF-β-induced EMT, and autocrine BMP signaling is decreased while TGF-β signaling is increased, when compared to normal epithelial cells [48▪▪]. Supporting these reports, BMP2 and BMP7 antagonize TGF-β-mediated EMT, decrease the population of breast CSCs, and inhibit bone metastasis after intracardiac injection of MDA-MB-231 breast cancer cells in Balb/c nu/nu mice . Moreover, in immortalized mammary epithelial cells, TGF-β induces the expression of the transcription factor Sox4, which is required for EMT, and, when overexpressed, induces TGF-β-dependent EMT .
The correlation of carcinoma cell plasticity through EMT with CSC properties may help explain the role of CSCs in multistep cancer progression. Indeed, oncogenic mutations that normally occur in differentiated cancer cells can integrate into CSCs following dedifferentiation through EMT, and these CSCs with new oncogenic mutations can then contribute to cancer progression toward metastasis . In this way, TGF-β-induced EMT promotes metastasis not only by enabling primary carcinoma cells to invade and disseminate, but also by generating cells with both a mesenchymal and a stem-cell-like phenotype that then can give rise to secondary tumors with epithelial characteristics, resembling the primary tumor (Fig. 2). This reversion to the differentiated epithelial-like phenotype is important for the formation of the bulk of the secondary tumor mass . This concept has been proposed as a migrating CSC model, in which CSCs that undergo EMT drive clonal expansion at sites of dissemination .
Several recent reports support a role for TGF-β in this model. In primary human ovarian cancer cells, TGF-β secreted in the tumor microenvironment induces the expression of tissue transglutaminase 2, which induces EMT and CSC properties to enhance metastasis . The metastatic regulator, Six1, increases the expression of miR-106b-25 cluster, which targets and inhibits Smad7 expression, to activate TGF-β signaling and induce EMT and a CSC phenotype in human breast cancer cells . Finally, epigenetic regulation of the gene encoding E-cadherin not only controls EMT, but also CSC properties. H3K9me2 methylation of the E-cadherin gene by G9a is required for metastasis and stem-cell-like properties of breast cancer cells [31▪]. These observations illustrate and emphasize the importance of reversible EMT induced by TGF-β in CSCs and cancer progression.
In contrast to these and other reports demonstrating that TGF-β signaling induces the expansion of the CSC population and promotes cancer progression, TGF-β has also been shown to decrease the number of CSCs and inhibit tumor formation in several types of carcinomas [70–72]. Therefore, the link between activation of EMT by TGF-β and the expansion of the CSC population may not be a general property of all carcinomas, and may be regulated by other signaling pathways.
EPITHELIAL–MESENCHYMAL TRANSITION IN TUMOR STROMAL CELLS
Functional interactions of cancer cells with stromal cells in the tumor microenvironment are important determinants for cancer progression. Most of the cells in the tumor microenvironment are fibroblasts, that is, CAFs, which express α-smooth muscle actin and closely resemble myofibroblasts, and these stromal fibroblasts promote cancer progression . TGF-β has been shown to stimulate the differentiation of mesenchymal precursors into myofibroblasts , and thus contributes to the generation of CAFs, providing a scenario for how TGF-β can contribute to cancer progression through effects on stromal fibroblasts. Autocrine TGF-β and SDF-1 signaling maintain the differentiation of human mammary fibroblasts into myofibroblasts during tumor progression . CAFs were shown to extensively derive from endothelial cells through endothelial–mesenchymal transition, a process similar to EMT . TGF-β has been shown to stimulate the generation of CAFs from endothelial cells.
The differentiation plasticity of carcinoma cells that results in the generation of CSCs from differentiated cancer cells may also enable the generation of cancer stromal cells from carcinoma cells. In fact, several recent reports demonstrate that glioblastoma stem cells can transdifferentiate into endothelial-like cells that then contribute to the tumor neovasculature [76,77,78▪▪,79▪▪]. These observations suggest that certain tumors may induce cancer cells to dedifferentiate and transdifferentiate to produce stromal cell types that in turn support maintenance and progression of cancer. Thus, EMT may convert carcinoma cells into mesenchymal, fibroblast-like cancer cells that may then serve as CAFs. In this way, TGF-β-induced EMT may further enhance tumor progression through the generation of CAFs from cancer cells, following EMT-mediated dedifferentiation and transdifferentiation (Fig. 2).
EMT is now considered to play fundamental roles in the initiation and progression of carcinomas. Epithelial cell plasticity manifested by TGF-β-induced EMT and its reversibility appears to play pivotal roles in the control of many aspects of cancer progression. The roles and molecular mechanisms of TGF-β signaling in EMT have been extensively studied, very often in cell culture, but also in mouse models. The recent development of novel techniques, including intravital imaging, is likely to extend our capabilities to elucidate the mechanisms that regulate epithelial plasticity in a physiological context and their roles in cancer progression. Together with molecular mechanistic studies, these observations will provide a better understanding of the roles of TGF-β signaling in the progression of carcinomas and lead to the development of new therapeutic or prognostic strategies for the treatment of cancers.
The authors apologize to those researchers whose work was not included in this review because of space limitation.
Conflicts of interest
There are no conflicts of interest.
Research in the laboratory of R. D. was supported by the NIH grants RO1 CA63101 and CA136690 to R. D. S. L. is supported by the American Heart Association scientist development award (grant number SDG2280008), and Y. K. is supported by a fellowship from the Cell and Science Research Foundation (Japan).
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Papers of particular interest, published within the annual period of review, have been highlighted as:
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Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 102).
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This study shows the evidence for the generation of stromal cells from CSCs. They describe the ability of glioblastoma stem cells to transdifferentiate into endothelial cells that support tumor growth and maintenance.
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