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Published ahead of print on February 16, 2006, doi:10.1165/rcmb.2006-0051SF
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American Journal of Respiratory Cell and Molecular Biology. Vol. 35, pp. 3-9, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2006-0051SF

Airway Epithelium, Inflammation, and Mechanisms of Disease: A Tribute to Carol B. Basbaum

Epithelial–Mesenchymal Transition in Lung Cancer

Is Tobacco the "Smoking Gun"?

Vijay Dasari, Marianne Gallup, Hassan Lemjabbar, Inna Maltseva and Nancy McNamara

Department of Anatomy, University of California, San Francisco, San Francisco; and Vision Science, University of California, Berkeley, Berkeley, California

Correspondence and requests for reprints should be addressed to Nancy McNamara, Department of Anatomy, Box 0452, 513 Parnassus, HSW 1330, University of California, San Francisco, San Francisco, CA 94143-0452. E-mail: nancy.mcnamara{at}ucsf.edu

Over the last several years, epithelial–mesenchymal transition (EMT), a process in which cells undergo a switch from an epithelial phenotype to a mesenchymal phenotype, has emerged as important not only in embryonic development, but also as a potential mechanism for cancer progression. Epithelial cells are characterized by cell–cell interactions, with the formation of tight junctions, lateral, apical, and basal membranes, polarized distribution of cellular components, and lack of mobility, whereas mesenchymal cells have loose interaction with other cells, are not polarized, and can be motile. In order for a cell to undergo EMT, a number of dramatic cellular and molecular changes must take place. These changes include: dissolution of adherens junctions, reorganization of the actin cytoskeleton, loss of apical–basal polarity, induction of promesenchymal gene expression, and migration through basement membranes and tissues. In this review, we describe the molecular components that drive the transformation of normal cells to tumor cells, with an emphasis on the complex signaling networks that underlie EMT. We then examine how each of these players might mediate the transformation of lung epithelial cells induced by tobacco smoke.

INTRODUCTION

There are many parallels between the process of epithelial–mesenchymal transition (EMT) in morphogenesis and the role of EMT in driving carcinogenesis. Some of these parallels include loss of E-cadherin expression and increased activation of tyrosine kinase receptors, such as the epidermal growth factor receptor (EGFR) (1), fibroblast growth factor receptor (FGFR) (2), and insulin-like growth factor receptor (IGFR) (3). Many other signaling pathways crucial for embryonic development, such as the Ras–mitogen-activated protein kinase (MAPK) pathway and Src, are also activated in tumor cells. During development, EMTs are tightly regulated and are required for morphogenetic movements underlying parietal endoderm formation and gastrulation, as well as the formation of a number of organs and tissues (4). In tumorigenesis, the highly regulated process of EMT becomes time-independent, and is characterized by selective amplification of particular aspects of the process while other events are bypassed (5). Amplification of only some of the full-fledged aspects of EMT in development is believed to drive preferential oncogenic pathway activation that leads to increased motility, invasion, and malignant transformation of cells (5, 6, reviewed in Ref. 7).

REACTIVE OXYGEN SPECIES

Reactive oxygen species (ROS) are oxygen species with unpaired electrons (free radicals), like hydrogen peroxide or superoxide anion. They are formed by several mechanisms. In the mammalian cell, they can be formed by ionizing radiation or produced as a by product of cellular respiration. They are also synthesized directly by enzymes such as reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or myeloperoxidase (in neutrophils). ROS can stimulate invasion of cells into the extracellular matrix (ECM) (8), induce matrix remodeling (9), effect cellular adhesion (10), and influence a number of signaling pathways (11). Our lab and others have reported the formation of ROS in lung epithelial cells after exposure to tobacco smoke (12, 13). Furthermore, a recent report demonstrated ROS-induced EMT in mouse mammary cells by MMP3 through increased transcription of Rac1b. This upregulation resulted in stimulation of Snail via ROS, an important repressor of E-cadherin (14). This evidence clearly points to the significance of ROS production by cigarette smoke and its potential induction of a cancerous phenotype in lung epithelial cells.

EGFR

EGFR is overexpressed in many types of cancers, including non–small cell lung cancer (NSCLC) (15). The presence of activating mutations in EGFR in tumors from patients with NSCLC, leading to resistance to treatment with EGFR-blocking drugs, was recently documented (16–19). Others have shown that mutations are not sufficient to cause resistance to these drugs (20). Using human tumor cells, Lu and colleagues (21) showed that chronic EGF treatment caused downregulation of caveolin-1 and subsequent induction of EMT through disruption of cell–cell adhesion, downregulation of E-cadherin expression via endocytosis, and induction of the transcriptional repressor Snail. It has now been shown that sensitivity to EGFR inhibition using erlotinib is directly linked to the degree of EMT in NSCLC tumors (22). It is clear from these data that EGFR overexpression or constitutive activation through mutations is an important aspect of the lung cell cancer phenotype.

Dr. Diazong Li, in our lab, found that smoke exposure of epithelial cells in culture led to the phosphorylation of EGFR. Subsequently, we found that this activation was induced through oxygen radicals, leading to the activation of TNF-{alpha}–converting enzyme, causing cleavage of the EGFR ligand amphiregulin (12). Further elucidation of this pathway and its importance for EMT is needed.

PHOSPHATIDYLINOSITOL 3-KINASE/SERINE–THREONINE PROTEIN KINASE B/GLYCOGEN SYNTHASE KINASE-3beta–/–

Src and Ras can induce EMT through phosphatidylinositol 3-kinase (PI3K)/serine–threonine protein kinase B (Akt) and MAPK pathways (4, 23). Akt is a serine–threonine kinase that, when activated, induces EMT through loss of cell adhesion, changes in morphology, loss of cell polarization, and increased cell motility (24). These pathways, together with wingless-type MMTV integration site family of proteins (Wnt) signaling (see below), inhibit glycogen synthase kinase (GSK)-3beta and trigger EMT via the stabilization and nuclear localization of the transcription factor Snail (25). Transcription of Snail has also been found to be stimulated by GSK-3 inhibition (26). Snail is a strong repressor of E-cadherin. Its overexpression leads to phenotypic changes that include increases in the mesenchymal markers, vimentin and fibronectin, as well as the acquisition of migration/invasion properties (27).

In the lung, several of these pathways have recently been linked to protumorigenic events induced by tobacco exposure. NF-{kappa}B activation induced by cigarette smoke condensate was completely suppressed in GSK-3beta–/– cells (28). Recent investigation found that murine lung lesions induced by the tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, had increased PI3K/Akt activity (29). The same group demonstrated that 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and nicotine activated the Akt pathway and increased proliferation in NSCLC cells (30).

TRANSFORMING GROWTH FACTOR-beta

Members of the transforming growth factor (TGF)-beta superfamily are multifunctional cytokines that control cell fates, including cell cycle arrest, differentiation, and apoptosis (31). Numerous studies have demonstrated the importance of TGF-beta in controlling epithelial cell plasticity, indicating that TGF-beta–dependent signals may play an important role in inducing EMT phenotypes. The complex function of this cytokine in transdifferentiation is related to the ability of TGF-beta isoforms to signal through a variety of molecules, such as Smads, RhoA GTPase, PI3K, and MAPK. Together, TGF-beta target molecules create an extensive network of interacting and independent signaling pathways (32). Key events in EMT are downregulation of epithelial-specific proteins (e.g., tight- and adherens-junction proteins), induction of various mesenchymal proteins (e.g., vimentin), and the ability to digest and migrate through the ECM (33). Recent investigations shed some light on the role of TGF-beta in each of these events.

Dissolution of tight junctions is an early event in EMT. Interaction of TGF-beta with its receptor leads to activation of protease activated receptor (PAR) 6 (34), a key regulator of epithelial cell polarity and tight-junction assembly (35). PAR6 is required for TGF-beta–dependent EMT and, upon phosphorylation, PAR6 interacts with the E3 ubiquitin ligase, Smurf1, and subsequent degradation of RhoA causes loss of tight junctions (36) and junctional stability (37). TGF-beta also induces disassembly and disappearance of E-cadherin–supported adherens junctions (38), further contributing to the loss of integrity of the epithelial barrier.

A switch to a highly motile phenotype marks further progression toward EMT. Remodeling of actin cytoskeleton by a change from cortical actin to stress fibers is a hallmark of a migratory, mesenchymal cell (39). TGF-beta–initiated activation of RhoA leads to contractility of cytoskeletal structures (40) and formation of focal adhesions of structural proteins and integrins, enabling cells to communicate with ECM (41). Upregulation of matrix metalloproteinase (MMP)-2 and MMP-9 by TGF-beta (42, 43) further promotes EMT via degradation of collagen type IV components of the basement membrane, aiding in epithelial unit dissociation.

Cigarette smoking is the major cause of lung cancer, and it is likely that smoke leads to disease by a variety of mechanisms. One potential mechanism may involve increased expression of RhoA GTPase and subsequent cytoskeletal remodeling. GTP-bound RhoA has been detected in the bronchial smooth muscle of rats that were exposed to cigarette smoke (44). We have also observed smoke-induced activation of RhoA in bronchial epithelial cells. Interestingly, this activation occurs in conjunction with increased cell motility (unpublished data). The importance of TGF-beta in smoke-induced lung disease is highlighted by a recent study demonstrating that cigarette smoke causes rapid release of active TGF-beta and subsequent autocrine downstream signaling (45). The same group demonstrated that TGF-beta production was increased in airway epithelial cells and suggested that ongoing cytokine production in chronic smokers may contribute to increased airway epithelial cell proliferation. Aberrant proliferation of airway epithelial cells has been observed in bronchial biopsies from both current and former smokers with chronic bronchitis (46). Taken together, these observations suggest that smoke might play a role in TGF-beta–mediated EMT.

DEVELOPMENTAL SIGNALING PATHWAYS

The "unscheduled" activation of embryonic signaling pathways that are necessary for stem cell function and development provide a major driving force for EMT and tumor growth. Examples of such pathways include Wnt/beta-catenin, Hedgehog (Hh), and Notch signaling. The overall function of each pathway in EMT has been studied extensively; however, the specific role of each in lung cancer is less clear.

E-Cadherin, Wnt/beta-Catenin, and Muc1
Cadherins comprise a large family of cell–cell adhesion molecules (47–49), and deregulation of cadherin-mediated adhesion can result in EMT and increased cell motility (23, 50). The extracellular domain of E-cadherin forms calcium-dependent homophilic interactions with neighboring cells, whereas the intracellular domain interacts with catenins (i.e., {alpha}-catenin, beta-catenin, and plakoglobin) to anchor the adhesion complex to the actin cytoskeleton (51). Recent studies have shown that several cytoplasmic signaling pathways are activated by cell–cell contact, and others are activated by loss of contact. For example, cadherin-mediated contacts regulate the availability of beta-catenin in Wnt signaling (reviewed in Ref. 52). beta-catenin has a dual role in epithelial cells, serving both to reinforce intercellular adherens junctions and, when released from these junctions, to enter the nucleus and modulate gene transcription via the Wnt signaling pathway.

Wnt signaling has been demonstrated in many cancers, and has recently emerged as a critical pathway in lung carcinogenesis (reviewed in Ref. 53). In the absence of Wnt signals, beta-catenin exists in a complex with APC and axin, and is targeted for ubiquitin/proteasome-mediated degradation after phosphorylation by GSK3beta. When Wnt is present, it binds to its receptor, Frizzled, and activates Disheveled. Disheveled blocks beta-catenin degradation, and excess beta-catenin enters the nucleus where, in association with the transcription factor T cell factor/lymphocyte enhancer factor (TCF/LEF), it turns on protumor gene expression (a list of Wnt-target genes is provided online at http://www-leland.stanford.edu/~rnusse/wntwindow.htm). One direct transcriptional target of beta-catenin/TCF is Slug, a zinc-finger protein shown to be involved in mesoderm formation (54). Another zinc-finger protein, Snail, is stabilized by Wnt signaling (55), and its expression is inversely correlated with E-cadherin transcription. Transcriptional repression of E-cadherin by Snail is closely correlated with EMT, and expression of Snail is induced by the integration of Wnt/beta-catenin signaling with the TGF-beta signaling pathway (reviewed above). beta-catenin–TCF/LEF complex formation induces the transcription of many other target genes that have been implicated in cancer. Our own RNA profiling studies have shown induction of several Wnt target genes in response to tobacco smoke. The role of Wnt signaling in lung cancer is also suggested by recent studies showing overexpression of Disheveled (56) and repression/silencing of Wnt antagonists, such as Wnt inhibitory factor (WIF)-1, in lung tumors and lung cancer cell lines (57, 58).

In lieu of a soluble Wnt ligand, Wnt/beta-catenin–dependent gene expression can be triggered by pathologic processes, such as junction disassembly. For example, the release of beta-catenin from disassembled adherens junctions between epithelial cells occurs after proteolytic destruction of E-cadherin by receptor tyrosine kinase (RTK)/Ras and TGF-beta signaling. This strongly enhances cytoplasmic beta-catenin and the transcription of TCF/LEF target genes. In this respect, the dissolution of junctions mimics effects of the embryonic Wnt signaling pathway. In our own studies with tobacco smoke, we have observed dissolution of adherens junctions and translocation of beta-catenin to the nucleus (unpublished data). Interestingly, this appears to occur in conjunction with an EGFR-dependent increase in the interaction between beta-catenin and the cytoplasmic tail of MUC1 (unpublished data).

MUC1 is a membrane-bound mucin that is expressed on the apical borders of epithelial cells (59). Upon transformation of epithelial cells, high levels of MUC1 become expressed over the entire cell surface (60). The cytoplasmic domain of MUC1 has been shown to interact with a number of proto-oncogenes, including EGFR, beta-catenin, and p120 catenin (61–64). Phosphorylation of MUC1 cytoplasmic tail by EGFR promotes its interaction with beta-catenin. Although the significance of this finding is currently unclear, we postulate that MUC1 contributes to EMT in the setting of smoke-induced malignant transformation by out competing E-cadherin for beta-catenin binding and escorting it to the nucleus, where it turns on Wnt target genes to promote tumorigenesis.

Hedgehog
Originally identified as a mediator of segment polarity in Drosophila (65), the Hh pathway is essential for normal embryonic development in mammals (66). The three mammalian homologs of the Drosophila gene Hh, Sonic (Shh), Indian Hh, and Desert Hh, establish morphogenic gradients essential for axial patterning in the mammalian embryo (66–68). Shh is the predominant signaling molecule in lung, brain, and limb development, and is the most extensively studied Hh protein in vertebrates (69–72).

The active 19 kD Shh ligand is lipid-modified at the N and C termini, (73, 74) and binds, in responsive cells, to the Hh receptor Patched (Ptch). Ptch is an unusual 12-transmembrane protein with homology to a bacterial transporter (75). In absence of Hh ligand, Ptch acts to inhibit the seven-transmembrane protein Smoothened (Smo), rendering downstream effectors of the pathway inactive (75). Binding of Hh ligand inactivates Ptch, derepressing Smo and resulting in positive Hh pathway signaling (66, 71, 76). In Drosophila, the effector component of Hh signaling is the transcription factor Cubitus interruptus (77–80) which is activated by Hh signaling through Smo. Mammalian homologs of Cubitus interruptus include three glioma-associated oncogene (Gli) proteins, which vary in their transcriptional activity (71, 81). The oncoprotein, Gli1, is a strong positive regulator of Hh pathway, whereas Gli2 and Gli3 possess both transcriptional activation and repression properties (72, 82–88). A large subpopulation of both lung (89) and pancreatic (90) cancer cell lines are dependent on Hh signaling pathways. Our unpublished data also indicate a role for Hh in smoke-induced lung cancer. Interestingly, we have found that Hh gene targets are upregulated by smoke, and smoke-induced tumors undergo degeneration in response to the Hh inhibitor, cyclopamine (unpublished data).

The importance of Hh signaling in cancer lies in the nature of the transcription targets of Gli. These include Gli itself, as well as genes known to control cell proliferation: cyclin D1, cyclin E1, and myelocytomatosis viral oncogene (Myc); Gli also transactivates genes known to control angiogenesis (components of vascular endothelial growth factor and platelet-derived growth factor signaling pathways; reviewed in Ref. 91). Although the mechanisms responsible for activating the Hh pathway in cancer cells are poorly understood, there is substantial evidence for the presence of activating mutations. These have been described in tumors of skin, brain, skeletal muscle, esophagus, stomach, and pancreas (90–92). Based on the current (and emerging) availability of drugs to inhibit Hh signaling, it is possible that an understanding of the role of Hh in lung cancer pathogenesis will permit new approaches to prevent and reverse this disease.

Notch
The Notch pathway demonstrates both tumor-promoting and tumor-suppressing functions. Notch-dependent upregulation of cell-cycle inhibitors and suppression of Wnt and Hh signaling represent tumor-suppressive functions that can be disrupted when oncogenes are present (reviewed in Ref. 93). Although it is well established that Notch receptors signal through the regulation of hairy enhancer of split (Hes) and Hes-related proteins, such as hairy/enhancer of split-related with YRPW Motif 1 (Hey1), to repress transcription, there is considerable crosstalk with other signaling pathways. For example, the presence of Notch ligands can be induced through EGF/FGF family activation of Ras or TGF-betaR induction of Smads. Increased cleavage/activation of Notch can also occur directly via RTKs. Depending on cell type, Notch signaling can upregulate Hey1 or Snail, both of which repress target genes, such as E-cadherin, by binding to E-boxes (as reviewed below). Thus, there is considerable cooperation between Notch, TGF-beta, and RTK signaling pathways, and the EMT promoting function of Notch depends on both cellular context and whether cooperating oncogenic pathways are activated (7).

Although the role of Notch in human lung cancer is still unclear, fetal lung developmental studies suggest that Notch signaling plays a critical role in regulating airway epithelial development (reviewed in Ref. 94). Notch components are expressed in the distal lung bud during branching morphogenesis, and animals bearing a Hes1 mutation display a hypotrophic phenotype. In the setting of tumorigenesis, recent work has shown that Notch3 is expressed in 39% of resected human lung tumors, and inhibition of Notch3-mediated signaling in vitro dramatically reduces anchorage-independent growth. Interestingly, effects mediated via Notch3 appear to occur downstream of EGF-dependent activation of a MAPK pathway (95). In agreement, NSCLCs, including adenocarcinoma, appear to actively use this conserved pathway (reviewed in Ref. 94). Although there is clearly evidence to support a role for Notch in both lung development and cancer, it is interesting to note that our own studies of smoke-induced malignant transformation did not uncover a functional role for Notch signaling.

OTHER EMT REGULATORS LINKED TO SMOKE

Apoptosis and NF-{kappa}B
Cigarette tar contains over 6,000 compounds, including nicotine, phenols, polycyclic aromatic hydrocarbons, and nitrosamines (96). Because many of these compounds are water soluble, there is an aqueous, nicotine-containing solution that comes into close contact with the epithelial cells in the lungs of smokers and passive smokers. Previous work from our laboratory has shown that cigarette smoke activates growth-promoting intracellular signaling pathways to facilitate the proliferation of lung epithelial cells (12). In conjunction with overactivity of growth-promoting agents, smoke-exposed cells are able to survive programmed cell death through inhibition of apoptosis. Apoptosis is initiated in a cell after DNA damage (reviewed in Ref. 97). To resist the apoptotic response, cells can overexpress antiapoptotic proteins, and thereby increase the ratio of anti to proapoptotic proteins. One of the key survival factors implicated in enhancing antiapoptotic genes is the transcription factor, NF-{kappa}B.

The NF-{kappa}B family of proteins is ubiquitously expressed, and includes p65 (RelA), p50, c-Rel, and RelB. These proteins form heterodimers or homodimers, with the predominant form of NF-{kappa}B being a p65/p50 heterodimer. The first indication that NF-{kappa}B suppresses apoptosis came from the analysis of RelA–/– mice, which died at Embryonic Day 15 as a result of extensive liver apoptosis (98). In particular, NF-{kappa}B is involved in the upregulation of antiapoptotic genes, such as Bcl2, Bcl-xL, Mcl-1, and inhibitor of apoptosis protein. We and others have shown increased expression of the antiapoptotic gene Bcl2 in response to cigarette smoke extract (unpublished data). This increase is a direct result of activation of NF-{kappa}B family members, p65 and p50.

Constitutive nuclear NF-{kappa}B activity is linked to human leukemias, lymphomas, and solid tumors. Furthermore, several oncoproteins, including Ha-Ras, are known to activate NF-{kappa}B to mediate its transforming activity (99). Although the exact role of NF-{kappa}B in the pathogenesis of human tumors remains to be determined, suppression of apoptosis is clearly of major importance.

The precise role of NF-{kappa}B in regulating invasive responses, such as EMT and metastasis, is poorly understood, but evidence suggests an important in vitro correlation between EMT and the enhanced expression/activation of NF-{kappa}B. For example, a recent report by Huber and colleagues demonstrates a central role for NF-{kappa}B in the induction of EMT (100). The authors showed that Ha-Ras–transformed mammary epithelial cells are prevented from progressing through EMT in response to TGF-beta when NF-{kappa}B activity is blocked. Moreover, activation of NF-{kappa}B promoted EMT even in the absence of TGF-beta. Blocking of NF-{kappa}B also reduced the metastatic potential of Ras-transformed mammary epithelial cells in vivo (100). Enhanced activity of NF-{kappa}B is also correlated with expression of the mesenchymal markers, vimentin and tenascin C. Taken together, these data provide evidence that NF-{kappa}B plays an essential role in both the induction and maintenance of EMT (7).

Loss of Cell Polarity
Loss of intercellular adhesion is a hallmark of EMT and a key step in the development of an aggressive tumor cell phenotype. Dissolution of the adherens junction can occur as a consequence of lost expression or transcriptional repression of E-cadherin (reviewed in Ref. 101). E-cadherin is regulated at both the mRNA and protein levels by means of changes in subcellular distribution, translation or transcriptional events, and degradation (5). Several other mechanisms have also been described that can suppress E-cadherin expression, including somatic mutations, promoter hypermethylation, and histone deacetylation. Because E-cadherin is considered to be a tumor suppressor, the identification of transcription factors that cause E-cadherin repression and EMT induction has been the topic of intensive investigation (reviewed in Refs. 5, 7, 101). As discussed above, prominent repressors of E-cadherin expression include the Snail-related zinc-finger transcription factors (Snail and Slug). Other transcriptional repressors include the ZEB-family, basic helix-loop-helix, E12/E47, Twist, and the list continues to grow. Snail, Slug, Zeb2, and Zeb1 bind to the consensus sequence, CANNTG, known as an E-box, and the human E-cadherin promoter contains three E-boxes. Another consensus binding site shown to downregulate E-cadherin expression is the Erythroblastosis virus E26 oncogene (Ets) site. The binding of Ets to the E-cadherin promoter (at position –97) has been shown to downregulate E-cadherin promoter activity in a keratinocyte cell line producing this protein (5, 102). Interestingly, Ets factors are also involved in the regulation of several key mediators of metastatic invasion, including matrilysin, MMPs, heparinase, and urokinase (reviewed in Ref. 103). Events leading to E-cadherin repression and EMT induction appear to occur via multiple signal transduction pathways, including those mediated via TGF-beta, Akt, Wnt, and Ras/MAPK. Many of these signaling pathways are activated in smoke-exposed epithelial cells. Perhaps as a consequence, we have observed that, in as few as 3 d of smoke exposure in vitro, airway epithelial cells separate from their neighbors and adopt pleiomorphic shapes suggestive of EMT (our unpublished data).

CONCLUSIONS

The role of EMT in cancer progression is a topic of debate in the scientific community that stems from a paucity of in vivo data demonstrating the importance of EMT in tumorigenesis (104, 105). Despite this ongoing discussion, it is clear that several of the key pathways driving EMT in the highly controlled process of development are also aberrantly activated in cancer. In this brief review, we explored the role of EMT and its possible induction by tobacco smoke. Both active and passive smoke exposure threatens the well-being of millions of Americans, and the molecular mechanisms underlying smoke-induced lung disease remain unclear. In the laboratory of Dr. Carol Basbaum, we have examined the direct effects of tobacco smoke on lung cell homeostasis, and have thereby defined protumor phenomena induced by smoke, including: loss of cell–cell adhesion, proliferation, inhibition of apoptosis, cell motility, and activation of embryonic signaling pathways. Further studies to elucidate the molecular mechanism(s) that underlie these events are currently in progress. A summary of the pathways thought to drive lung tumorigenesis in response to smoke is shown in Figure 1. Studies directed at defining the key molecular components of each pathway may provide novel drug targets that can be used to direct therapies toward the prevention and/or treatment of lung cancer.


Figure 1
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  		Figure 1. Signaling diagram: EMT and smoke. Four main signaling pathways activated by smoke exposure may contribute to EMT. Shh is a ligand that binds to its receptor, Ptch. Binding of Hh inactivates Ptch and derepresses the transmembrane protein, Smo, resulting in positive Hh signaling. This includes DNA binding of the transcription factor Gli, and activation of its target genes, such as cyclin D and Myc. Wnt is another ligand that, when bound to its receptor, Frizzled (Fz), leads to inactivation of GSK3. This prevents proteosomal degradation of beta-catenin, and leads to the translocation of beta-catenin into the nucleus, where it increases protumor gene expression through complex formation with the transcription factor TCF/LEF1. In lieu of a soluble Wnt ligand, Wnt/beta-catenin gene expression can be triggered by the release of beta-catenin from disassembled junctions. Smoke-inducing interactions between beta-catenin and the membrane glycoprotein, MUC1, appear to promote junction disassembly by out competing beta-catenin for E-cadherin. ROS are produced through the activation of NADPH oxidase, and lead to the activation of the transcription factor NK-{kappa}B. The activation of NK-{kappa}B leads to decreased expression of E-cadherin through the activation of Snail and upregulated expression of Bcl2, an inhibitor of apoptosis. Smoke-induced ROS also activates the A disintegrin and metalloprotease (ADAM), TNF-{alpha}–converting enzyme, to cause cleavage of amphiregulin, a ligand for EGFR. Activation of EGFR leads to Ras/Raf/MAPK, PI3K/Akt, and Src signaling. TGF-beta isoforms signal through Smads, RhoA, PI3K, and MAPK. TGF-beta leads to activation of PAR6, with the subsequent loss of tight junctions through the degradation of RhoA. TGF-beta–initiated activation of RhoA leads to cytoskeletal changes and increased migration.

 
Footnotes

Authors are listed in alphabetical order.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0051SF on February 16, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 1, 2006

Accepted in final form February 6, 2006

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