The role of epithelial plasticity in normal embryogenesis and in carcinoma invasiveness and metastasis is a topic of intense current research (Condeelis and Segall, 2003
; Grünert et al., 2003
). This emphasizes the need for identification of factors that mediate cancer metastasis. EMT characterizes changes in epithelial plasticity and probably represents a transitory stage of carcinoma cell evolution toward aggressive malignancy (Radisky et al., 2001
; Thiery, 2002
; Grünert et al., 2003
). Changes in epithelial plasticity during carcinoma progression are controlled by epigenetic, environmental, and cell communication mechanisms (Radisky et al., 2001
). In this context, secreted growth factors are of paramount importance, and the TGF-β superfamily includes many members that contribute to regulation of epithelial plasticity and growth (ten Dijke et al., 2002
; Siegel and Massagué, 2003
). One major task in the analysis of the TGF-β pathway is therefore the molecular dissection of components that contribute to processes such as cell cycle arrest or EMT.
Here we provide a comprehensive analysis of most components of the TGF-β superfamily signaling pathways using appropriate model cell systems and furthermore identify a group of novel gene targets of this pathway that potentially link basic TGF-β biology to epithelial cell invasiveness. A major conclusion is that EMT is a physiological response of both normal mouse and human epithelial cells to TGF-β. We observed a more robust scattering and depolarization in mammary and lung epithelial cells than in keratinocytes (), which may reflect the developmental history of each tissue type. Despite this, all cell types tested here, as well as several others such as kidney, prostate or lens epithelial cells, undergo EMT (reviewed in Gotzmann et al., 2004
). The observations in normal keratinocytes are in agreement with another report, which analyzed HaCaT cell EMT in response to TGF-β1 (Zavadil et al., 2001
). Malignant keratinocytes expressing Ras oncogenes exhibit stronger EMT and architectural depolarization, which is mediated by TGF-β and appears more similar to the response described here for normal mammary cells (Portella et al., 1998
). The data also suggest that TGF-β is sufficient to elicit a transient but fully developed EMT response in normal epithelial cells without the need for predisposing oncogenic transformation as documented for the Ras
oncogene (Janda et al., 2002
; Oft et al., 2002
; Grünert et al., 2003
). We also demonstrate that the in vitro EMT response of normal epithelial cells is conserved between mice and humans. Our data suggest that NMuMG cells represent a faithful in vitro cell model of EMT despite the fact that these cells carry no autonomous tumorigenic potential.
Although the role of TGF-β on modulation of epithelial plasticity and tumor progression has become a major point of current research (ten Dijke et al., 2002
; Siegel and Massagué, 2003
), the contribution of the other members of the TGF-β superfamily to such processes remains poorly studied. Using adenovirus-mediated expression of all type I receptors of the superfamily we could delineate for the first time that only signaling pathways that belong to the TGF-β/activin/nodal branch can induce EMT (, Supplementary Figure 1). The low-molecular-weight inhibitor of the three receptor kinases ALK-4, -5, -7, gave evidence for constitutive autocrine TGF-β signaling that maintains low level gene expression in NMuMG cells (). Although all three ALK-4(TD), -5(TD), and -7(TD) receptors induced EMT in NMuMG cells, only ALK-5(KR) (and partially ALK-4(KR)) were able to block TGF-β1-induced EMT (). This reflects the fact that all three constitutively active receptors can activate robust Smad2/Smad3 signaling, whereas only ALK-5(KR) can efficiently interfere with the function of the endogenous TGF-β receptor complex. Because overexpressed activin type I receptor (ALK-4) is able to elicit EMT, we conclude that the failure of activin-A to induce EMT and Smad2 phosphorylation in NMuMG cells may reflect a suboptimal level of ALK-4 on the cell surface in this cell type.
A corollary from the comprehensive receptor analysis was that specific R-Smads, i.e., Smad2 and Smad3, might be involved in the process of EMT. Indeed, we previously reported a role for Smad3 downstream of TGF-β in NMuMG cell responses (Piek et al., 1999b
). We extended our analysis to essentially all Smad components of the superfamily, Smad1–7, with the sole exception of Smad8 for technical reasons (, Supplementary Figures 4–7). The new results strongly implicate the endogenous Smads of the TGF-β branch in signal transduction that orchestrates the EMT response. However, a previous report that used stably transfected NMuMG clones with wild-type Smad7 or a different dominant negative Smad3 mutant than Smad3(D407E), failed to observe effects on EMT in response to TGF-β (Bhowmick et al., 2001a
). In our hands it has been impossible to obtain stable clones of epithelial cells that express wild-type or mutant Smads constitutively (unpublished results). We either had to rely on inducible expression systems () or on transient adenoviral infections (). Thus, it is possible that NMuMG clones selected for survival in the continuous presence of high levels of exogenous mutant Smad3 or Smad7 might adapt by exhibiting differential responses to TGF-β. The data described here are in good agreement with several recent reports on the role of Smad4 in mammary EMT and on the ability of transcription factors YY1 and c-ski, which interact and inactivate Smad protein function, to block specifically EMT of NMuMG cells (Kurisaki et al., 2003
; Li et al., 2003
; Takeda et al., 2004
). The dominant-negative Smad2 and Smad3 mutants used unfortunately have not allowed us to pinpoint possible differential effects between the two R-Smads of the TGF-β pathway. This is in part due to the fact that these mutants interfere at least partially with both Smad2 and Smad3 activation by TGF-β receptors. This has been previously established for mutant Smad3(D407E) (Goto et al., 1998
) but not for mutant Smad2SA. In the stable clones expressing Smad2SA we could measure robust phospho-Smad3 levels induced by TGF-β1 (Supplementary Figure 4F). However, this finding does not necessarily exclude the possibility that the mutant interferes with other critical signaling effects, e.g., proper receptor-Smad endocytosis and trafficking. The fact that ectopic expression of Smad2 (or Smad3) alone could not elicit robust EMT in NMuMG cells (, Supplementary Figure 7) suggests that the Smad pathway as a whole is needed for this response, in addition to the cooperating non-Smad effectors.
The evidence supporting participation of Smads in the establishment of EMT by TGF-β is also corroborated by the analysis of the ALK-5 receptor with mutations in its L45 loop (), a result in full agreement with previous reports that examined the role of the L45 loop of ALK-5 on EMT of NMuMG cells (Yu et al., 2002
; Itoh et al., 2003
). Despite this, other evidence has supported the role of alternative signaling pathways in the same phenotypic response (Bakin et al., 2000
; Bhowmick et al., 2001a
; Yu et al., 2002
). Our evidence suggests that modulation of epithelial plasticity may be a more complex phenomenon than previously appreciated, in which both Smad and non-Smad effectors cooperate in order to execute a complete program of EMT. This would be consistent with the fact that EMT incorporates an extensive program of changes in gene expression (see below), part of which at least depends on Smad transcriptional inputs. Another part of this program may require the non-Smad signaling pathways, and a challenge for the immediate future remains to establish the points of cross-talk between the various effectors.
A remaining question concerned the types and numbers of genes regulated by TGF-β during the course of EMT. Transcriptomic analysis gave a rather rich outcome of regulated genes (, , and Supplementary Tables 3 and 4). This was anticipated based on recent transcriptomic screens of the TGF-β pathway in various cell types (Perou et al., 1999
; Akiyoshi et al., 2001
; Verrecchia et al., 2001
; Zavadil et al., 2001
; Ota et al., 2002
; Chambers et al., 2003
; Coyle et al., 2003
; Jechlinger et al., 2003
; Kang et al., 2003a
; Xie et al., 2003
; Kowanetz et al., 2004
). On the other hand, the gene list identified here shares relatively small similarities (only 22 common genes in contrast to 163 unique genes) with other transcriptomic analyses of the TGF-β pathway, including one recently reported in the same cell line (Xie et al., 2003
In our effort to define genes whose expression links to the EMT process, analyzing both early and late gene responses was instrumental. Thus, we were able to define gene clusters based strictly on expression values, which include genes with kinetics that suit with the onset and establishment of EMT in NMuMG cells (clusters 3 and 4, ). Combining this information with systematic screens of PubMed and cross-referencing with microarray data from studies of breast cancer, epithelial invasiveness, and metastasis (Clark et al., 2000
; Ryu et al., 2001
; van't Veer et al., 2002
; Jechlinger et al., 2003
; Kang et al., 2003b
), we selected a group of genes that possibly links to the EMT process. A number of these genes, such as Myh9, Cfh, Hif1a, Pdgfa, Ext1, Col6a1, Fn1, Sdc1, Gas1, Igfbp5, Iqgap1, Idb2, Cks-2, Vamp8,
have already been enlisted as poor prognosis markers for breast cancer or invasion and metastasis markers, but most of them have not been previously recognized as TGF-β responsive genes. Thus, we conclude that the NMuMG in vitro system, although more distant from mammary carcinoma cells undergoing EMT in vivo, represents a useful tool for the identification of candidate genes of EMT. Future work will focus on the actual in vivo validation of the novel TGF-β target genes.
To evaluate functionally some of the genes identified in this screen, we made use of the results on signaling specificity obtained in the first part of this study. We postulated that genes that are differentially regulated by TGF-β1 versus BMP-7 should be relevant targets that characterize the EMT process and can be used for future in vivo validation. Furthermore the newly established role of endogenous Smads on mammary EMT suggested that relevant gene targets might require an intact Smad pathway for proper regulation. Finally, we reasoned that critical TGF-β targets for regulation of EMT should exhibit conservation of their expression pattern among species. In agreement with our postulates, all genes tested showed a strict requirement for R-Smad activation by the TGF-β type I receptor (). In addition, most of the genes tested were not regulated by BMP-7 (), or, as is the case of the transcriptional regulators of the Id family, they exhibited the inverse response to BMP-7. Analysis of functional inactivation of Id2 using siRNA technology established a causal relationship between BMP-7 signaling and proper regulation of the myoepithelial marker α-SMA (). This is in perfect agreement with the model we have recently established, based on which, arrest of epithelial proliferation and induction of EMT require the down-regulation of Id2 (and Id3) by TGF-β. On the other hand, BMPs that induce high levels of Id2 (or Id3) in epithelial cells fail to exhibit robust growth inhibition or EMT because of the inhibitory role Id proteins play in these processes (Kowanetz et al., 2004
). Examination of the pattern of regulation of the selected genes in HMEC and HaCaT cells (, ) revealed that a number of the selected genes showed cell type specificity and evolutionary conservation in terms of their expression and mode of regulation.
In conclusion, we show that epithelial-mesenchymal transition is a multigenic cellular response to TGF-β superfamily members. Among such members, TGF-βs, activins and possibly other ligands such as nodals, which have the common feature of activating Smad2 and Smad3, appear to be competent to elicit EMT, whereas other members cannot. The combined signaling specificity and transcriptomic analysis provides a first degree of molecular dissection of novel gene functions that link and orchestrate the differentiative programs underlying epithelial plasticity.