Dynamics of TGF-β induced epithelial-to-mesenchymal transition monitored by electric cell-substrate impedance sensing

The epithelial-to-mesenchymal transition (EMT) is a program of cellular development associated with loss of cell-cell contacts, a decreased cell adhesion and substantial morphological changes. Besides its importance for numerous developmental processes, EMT has also been held responsible for the development and progression of tumors and formation of metastases. The influence of the cytokine transforming growth factor β1 (TGF-β1) induced EMT on structure, migration, cytoskeletal dynamics and long-term correlations of the mammalian epithelial cell lines NMuMG, A549 and MDA-MB231 was investigated with time-resolved impedance analysis. The three cell lines show important differences in concentration dependency, cellular morphology and dynamics upon their response to TGF-β1. A549 cells and the non-tumor mouse epithelial cell line NMuMG show a substantial change in morphology mirrored in stepwise changes of their phenotype upon cytokine treatment. Impedance based measurements of micromotility reveal a complex dynamic response to TGF-β1 exposure which leads to a transient increase in fluctuation amplitude and long-term correlation. These changes in fluctuation amplitude are also detectable for MDA-MB231 cells, whereas the long-term correlation remains unvaried. We were able to distinguish three time domains during EMT. Initially, all cell lines display an increase in micromotion lasting 4 to 9h termed transitional state I. This regime is followed by transitional state II lasting approximately 20 h, where cellular dynamics are diminished and, in case of the NMuMG cell line, a loss of cell-cell contacts occurs. Finally, the transformation into the mesenchymal-like phenotype occurs 24-30 h after exposure to TGF-β1.     

EMT changes actin cortex rheology in a cell-cycle-dependent manner

The actin cortex is a key structure for cellular mechanics and cellular migration. Accordingly, cancer cells were shown to change their actin cytoskeleton and their mechanical properties in correlation with different degrees of malignancy and metastatic potential. Epithelial-mesenchymal transition (EMT) is a cellular transformation associated with cancer progression and malignancy. To date, a detailed study of the effects of EMT on the frequency-dependent viscoelastic mechanics of the actin cortex is still lacking. In this work, we have used an established atomic force microscope-based method of cell confinement to quantify the rheology of the actin cortex of human breast, lung, and prostate epithelial cells before and after EMT in a frequency range of 0.02–2 Hz. Interestingly, we find for all cell lines opposite EMT-induced changes in interphase and mitosis; whereas the actin cortex softens upon EMT in interphase, the cortex stiffens in mitosis. Our rheological data can be accounted for by a rheological model with a characteristic timescale of slowest relaxation. In conclusion, our study discloses a consistent rheological trend induced by EMT in human cells of diverse tissue origin, reflecting major structural changes of the actin cytoskeleton upon EMT.     

Inhibition of actin dynamics during epithelial-to-mesenchymal transition

Transforming growth factor β1 is one of the main inducers of epithelial-to-mesenchymal transition (EMT). During EMT cells from an ordered epithelial state adopt a fibroblast-like shape combined with a reorganization of the cytoskeleton and altered cell-cell and cell-substrate interactions. Interestingly, an increased cellular motion lasting up to 9h after cytokine stimulation takes place. These changes in cellular shape and dynamics can be monitored by impedance spectroscopy. Analyzing impedance noise by means of variance and detrended fluctuation analysis provides information about the magnitude of vertical cellular micromotility and the long-term correlation of the impedance signal. Via preincubation with Rho kinase inhibitor Y-27632, blebbistatin, and the protein inhibitors rapamycin and cycloheximide before cytokine addition, we were able to assign the origin of the dynamic changes. Fluctuations upon TGF-β1 administration were diminished using cycloheximide, blebbistatin and rapamycin. Consequently, we conclude that mainly actin contractility and de novo protein synthesis leading to changes in actin polymerization/depolymerization processes are responsible for the detected alterations, whereas activation of Rho kinases (ROCK) is not involved. Importantly, none of the used agents affected the EMT phenotype, reflected in unchanged static impedance parameters, optical micrographs and unmodified correlations displayed in the impedance noise.     

Glycoproteomic analysis of two mouse mammary cell lines during transforming growth factor (TGF)-β induced epithelial to mesenchymal transition

Background  

TGF-β acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. However, later in tumor development TGF-β can become tumor promoting through mechanisms including the induction of epithelial-to-mesenchymal transition (EMT), a process that is thought to contribute to tumor progression, invasion and metastasis. To identify EMT-related breast cancer therapeutic targets and biomarkers, we have used two proteomic approaches to find proteins that change in abundance upon the induction of EMT by TGF-β in two mouse mammary epithelial cell lines, NMuMG and BRI-JM01.     

Caveolin-1 up-regulation during epithelial to mesenchymal transition is mediated by focal adhesion kinase

Emerging evidence has shown that caveolin-1 is up-regulated in a number of metastatic cancers and can influence various aspects of cell migration. However, in general, the role of caveolin-1 in cancer progression is poorly understood. In the present study, we examined alterations in caveolin-1 expression during epithelial-to-mesenchymal transition (EMT) and the ability of caveolin-1 to alter cancer cell adhesion, an aspect of cell motility. We employed two EMT cell models, the human embryonic carcinoma cell line NT2/D1, and TGF-beta1-treated NMuMG cells, which are derived from normal mouse mammary epithelia. Caveolin-1 expression was substantially up-regulated in both cell lines following the induction of EMT and was preceded by increased activation of focal adhesion kinase (FAK) and Src, two known tyrosine kinases involved in EMT. We hypothesized that caveolin-1 expression could be influenced by increased FAK phosphorylation, to which Src is a known contributor. Examination of FAK+/+ and FAK-/- mouse embryonic fibroblasts revealed that in cells devoid of FAK, caveolin-1 expression is strikingly diminished. Using FAK and superFAK constructs and the novel FAK inhibitor PF-228, we were able to demonstrate that indeed, FAK can regulate caveolin-1 expression. We also found that Src can contribute to increases in caveolin-1 expression, however, only in the presence of FAK. From the culmination of this data and our functional analyses, we conclude that caveolin-1 expression can be up-regulated during EMT, and further, once expressed, caveolin-1 can greatly influence cancer cell adhesion.     

Dioxin Receptor Expression Inhibits Basal and Transforming Growth Factor β-induced Epithelial-to-mesenchymal Transition

Recent studies have emphasized the role of the dioxin receptor (AhR) in maintaining cell morphology, adhesion, and migration. These novel AhR functions depend on the cell phenotype, and although AhR expression maintains mesenchymal fibroblasts migration, it inhibits keratinocytes motility. These observations prompted us to investigate whether AhR modulates the epithelial-to-mesenchymal transition (EMT). For this, we have used primary AhR^+/+ and AhR^−/− keratinocytes and NMuMG cells engineered to knock down AhR levels (sh-AhR) or to express a constitutively active receptor (CA-AhR). Both AhR^−/− keratinocytes and sh-AhR NMuMG cells had increased migration, reduced levels of epithelial markers E-cadherin and β-catenin, and increased expression of mesenchymal markers Snail, Slug/Snai2, vimentin, fibronectin, and α-smooth muscle actin. Consistently, AhR^+/+ and CA-AhR NMuMG cells had reduced migration and enhanced expression of epithelial markers. AhR activation by the agonist FICZ (6-formylindolo[3,2-b]carbazole) inhibited NMuMG migration, whereas the antagonist α-naphthoflavone induced migration as did AhR knockdown. Exogenous TGFβ exacerbated the promigratory mesenchymal phenotype in both AhR-expressing and AhR-depleted cells, although the effects on the latter were more pronounced. Rescuing AhR expression in sh-AhR cells reduced Snail and Slug/Snai2 levels and cell migration and restored E-cadherin levels. Interference of AhR in human HaCaT cells further supported its role in EMT. Interestingly, co-immunoprecipitation and immunofluorescence assays showed that AhR associates in common protein complexes with E-cadherin and β-catenin, suggesting the implication of AhR in cell-cell adhesion. Thus, basal or TGFβ-induced AhR down-modulation could be relevant in the acquisition of a motile EMT phenotype in both normal and transformed epithelial cells.     

Cortical Mechanics and Meiosis II Completion in Mammalian Oocytes Are Mediated by Myosin-II and Ezrin-Radixin-Moesin (ERM) Proteins

Cell division is inherently mechanical, with cell mechanics being a critical determinant governing the cell shape changes that accompany progression through the cell cycle. The mechanical properties of symmetrically dividing mitotic cells have been well characterized, whereas the contribution of cellular mechanics to the strikingly asymmetric divisions of female meiosis is very poorly understood. Progression of the mammalian oocyte through meiosis involves remodeling of the cortex and proper orientation of the meiotic spindle, and thus we hypothesized that cortical tension and stiffness would change through meiotic maturation and fertilization to facilitate and/or direct cellular remodeling. This work shows that tension in mouse oocytes drops about sixfold during meiotic maturation from prophase I to metaphase II and then increases ∼1.6-fold upon fertilization. The metaphase II egg is polarized, with tension differing ∼2.5-fold between the cortex over the meiotic spindle and the opposite cortex, suggesting that meiotic maturation is accompanied by assembly of a cortical domain with stiffer mechanics as part of the process to achieve asymmetric cytokinesis. We further demonstrate that actin, myosin-II, and the ERM (Ezrin/Radixin/Moesin) family of proteins are enriched in complementary cortical domains and mediate cellular mechanics in mammalian eggs. Manipulation of actin, myosin-II, and ERM function alters tension levels and also is associated with dramatic spindle abnormalities with completion of meiosis II after fertilization. Thus, myosin-II and ERM proteins modulate mechanical properties in oocytes, contributing to cell polarity and to completion of meiosis.     

The cytoskeletal mechanics of brain morphogenesis

There is a functional device in embryonic ectodermal cells that we propose causes them to differentiate into either neuroepithelial or epidermal tissue during the process called primary neural induction. We call this apparatus the “cell state splitter”. Its main components are the apical microfilament ring and the coplanar apical mat of microtubules, which exert forces in opposite radial directions. We analyze the mechanical interaction between these cytoskeletal components and show that they are in anunstable mechanical equilibrium. The role of the cell state splitter is thus to create a mechanical instability corresponding to the embryonic state of “competence” in an otherwise mechanically stable cell. When the equilibrium of the cell state splitter is disturbed so as to produce a slight contraction of the apical end, apical contraction continues and the distinctive columnar neuroepithelial cells are produced. A slight expansion from the equilibrium state, on the other hand, results in flattened epidermal cells. The calculated forces are consistent with the know constitutive and force-generating properties and morphology of microfilaments and microtubules, and with free tubulin concentration. There are no free parameters in the analysis. The first cells to assume the neuroepithelial state lie over the notochord. Propagation of the neuroepithelial state (homoiogenetic induction) then proceeds via stretch-induced constriction of the apical microfilament rings, until ahemisphere is covered, at which point the high rate of change of the meridional stress component necessary for further propagation vanishes. The remaining cells are stretched somewhat by this process and become epidermis. A sharp boundary between the tissues is thus formed (explaining “compartmentalization” and the binary nature of differentiation in general). Normal induction apparently involves setup of the cell state splitters in all of the ectoderm cells, perhaps synchronously timed by global embryo tension. The initial transition of cells from the ectodermal to the neuroepithelial state begins at the notoplate, where cell attachments to the notochord may both cause basal actin deposition and significantly reduce the stress induced in the ectoderm by the global tension, biasing the notoplate cell state splitters toward the neuroepithelial state. Introduction of an organizer or other solid substrate (artificial inducer) elsewhere, to which ectodermal cells can adhere, may likewise have both of these effects. Differentiation to either epidermis or neuroepithelium is thus a machanical eventfollowed by the synthesis of specific proteins. This model of differentiation suggests that the genome responds to, rather than directly causes, differentiation.     

Epithelial-to-mesenchymal transition confers resistance to apoptosis in three murine mammary epithelial cell lines

Epithelial-to-mesenchymal transition (EMT) is an essential embryogenic and developmental process, characterized by altered cellular morphology, loss of cell adhesion, and gain of migratory ability. Dysregulation of this process has been implicated in tumorigenesis, mediating the acquisition of migratory and invasive phenotypes by tumor cells. Mammary epithelial cells provide an excellent model in which to study the process, being derived from mammary gland tissue that utilizes EMT to facilitate branching morphogenesis through which the developing gland migrates into and invades the fat pad. Inappropriate EMT has been heavily implicated in the progression of ductal hyperplasia and mammary tumor metastasis. We examined the morphological and molecular changes of three murine mammary epithelial cell lines following EMT induction. EMT was induced in the EpH-4 and NMuMG cell lines by transforming growth factor (TGF)-beta1 but not by ethanol, while the KIM-2 cell line was partially resistant to TGF-beta1 but responded fully to ethanol. The response to EMT-inducing reagent was shown to be critically dependent on the time of treatment, with confluent cells failing to respond. Timelapse photography identified increased motility during wound healing in cells pre-treated with EMT-inducing reagent compared with untreated controls. Furthermore, EMT conferred resistance to UV-induced apoptosis. Our data indicate that evaluation of characteristics other than loss and gain of phenotypic markers may be of benefit when assessing EMT, and contribute to the evidence suggesting that inappropriate EMT facilitates the acquisition of resistance to apoptosis, a key characteristic required for tumor survival.     

Hic-5 contributes to epithelial-mesenchymal transformation through a RhoA/ROCK-dependent pathway

Epithelial-mesenchymal transformation (EMT) in response to TGFbeta1 is a coordinated process of tissue morphogenesis that occurs during embryonic development as well as during certain pathologic events including kidney tubulointerstitial fibrosis. It is characterized by the disassembly of cell-cell junctions and dramatic alterations in the actin cytoskeleton that facilitates cell-matrix adhesion and stimulates migration. The focal adhesion adapter protein, Hic-5, has previously been reported to be upregulated during TGFbeta1-induced EMT in mouse mammary epithelial cells and the current study recapitulates this result in both mouse kidney proximal tubule epithelial, MCT, cells and human mammary epithelial, MCF10A, cells. To evaluate a causative role for Hic-5 in EMT, Hic-5 RNA interference (siRNA) was used to prevent Hic-5 expression in response to TGFbeta1 stimulation and was shown to suppress cell migration and actin stress fiber formation. It also resulted in the retention of a robust epithelial cell morphology characterized by elevated E-cadherin protein expression and well-organized adherens junctions. In addition, Hic-5 siRNA treatment led to the suppression of TGFbeta1 induction of RhoA activation. In contrast, forced expression of Hic-5 led to the formation of ROCK-dependent actin stress fibers. Furthermore, the induction of Hic-5 expression in response to TGFbeta1 was shown to be a RhoA/ROCK I-dependent process. Together, these data implicate Hic-5 as a key regulator of EMT and suggest that RhoA stimulated Hic-5 expression in response to TGFbeta1 may be functioning in a feed forward mechanism whereby Hic-5 maintains the mesenchymal phenotype through sustained RhoA activation and signaling.     

A Reaction-Diffusion Model of the Cadherin-Catenin System: A Possible Mechanism for Contact Inhibition and Implications for Tumorigenesis

Contact inhibition is the process by which cells switch from a motile growing state to a passive and stabilized state upon touching their neighbors. When two cells touch, an adhesion link is created between them by means of transmembrane E-cadherin proteins. Simultaneously, their actin filaments stop polymerizing in the direction perpendicular to the membrane and reorganize to create an apical belt that colocalizes with the adhesion links. Here, we propose a detailed quantitative model of the role of cytoplasmic β-catenin and α-catenin proteins in this process, treated as a reaction-diffusion system. Upon cell-cell contact the concentration in α-catenin dimers increases, inhibiting actin branching and thereby reducing cellular motility and expansion pressure. This model provides a mechanism for contact inhibition that could explain previously unrelated experimental findings on the role played by E-cadherin, β-catenin, and α-catenin in the cellular phenotype and in tumorigenesis. In particular, we address the effect of a knockout of the adenomatous polyposis coli tumor suppressor gene. Potential direct tests of our model are discussed.     

Actin bodies in yeast quiescent cells: an immediately available actin reserve?

Most eukaryotic cells spend most of their life in a quiescent state, poised to respond to specific signals to proliferate. In Saccharomyces cerevisiae, entry into and exit from quiescence are dependent only on the availability of nutrients in the environment. The transition from quiescence to proliferation requires not only drastic metabolic changes but also a complete remodeling of various cellular structures. Here, we describe an actin cytoskeleton organization specific of the yeast quiescent state. When cells cease to divide, actin is reorganized into structures that we named “actin bodies.” We show that actin bodies contain F-actin and several actin-binding proteins such as fimbrin and capping protein. Furthermore, by contrast to actin patches or cables, actin bodies are mostly immobile, and we could not detect any actin filament turnover. Finally, we show that upon cells refeeding, actin bodies rapidly disappear and actin cables and patches can be assembled in the absence of de novo protein synthesis. This led us to propose that actin bodies are a reserve of actin that can be immediately mobilized for actin cables and patches formation upon reentry into a proliferation cycle.     

A mitochondrial thioredoxin-sensitive mechanism regulates TGF-β-mediated gene expression associated with epithelial–mesenchymal transition

Transforming growth factor (TGF)-β is a pro-oncogenic cytokine that induces the epithelial–mesenchymal transition (EMT), a crucial event in tumor progression. During TGF-β-mediated EMT in NMuMG mouse mammary epithelial cells, we observed sustained increases in reactive oxygen species (ROS) levels in the cytoplasm and mitochondria with a concomitant decrease in mitochondrial membrane potential and intracellular glutathione levels. In pseudo ρ0 cells, whose respiratory chain function was impaired, the increase in intracellular ROS levels was abrogated, suggesting an important role of mitochondrial activity as a trigger for TGF-β-stimulated ROS generation. In line with this, TGF-β-mediated expression of the EMT marker fibronectin was inhibited not only by chemicals that interfere with ROS signaling but also by exogenously expressed mitochondrial thioredoxin (TXN2) independent of Smad signaling. Of note, TGF-β-mediated induction of HMGA2, a central mediator of EMT and metastatic progression, was similarly impaired by TXN2 expression, revealing a novel mechanism involving a thiol oxidation reaction in mitochondria, which regulates TGF-β-mediated gene expression associated with EMT.     

Morphological and mechanical stability of bladder cancer cells in response to substrate rigidity

BackgroundMorphology of cells can be considered as an interplay between the accessibility of substrate anchoring sites, cytoskeleton properties and cellular deformability. To withstand tension induced by cell's environment, cells tend to spread out and, simultaneously, to remodel actin filament organization.MethodsIn this context, the use of polyacrylamide hydrogel substrates with a surface coated with laminin allows to trace remodeling of actin cytoskeleton during the interaction of cells with laminin-rich basement membrane. Reorganization of actin cortex can be quantified by a surface spreading area and deformability of single cells.ResultsIn our study, we demonstrated that morphological and mechanical alterations of bladder cancer cells in response to altered microenvironment stiffness are of biphasic nature. Threshold-dependent relations are induced by mechanical properties of cell microenvironment. Initially, fast alterations in cellular capability to spread and to deform are followed by slow-rate changes. A switch provided by cellular deformability threshold, in the case of non-malignant cells, triggers the formation of thick actin bundles accompanied by matured focal adhesions. For cancer cells, cell spreading and deformability thresholds switch between slow and fast rate of changes with weak reorganization of actin filaments and focal adhesions formation.ConclusionsThe presence of transition region enables the cells to achieve a morphological and mechanical stability, which together with altered expression of vinculin and integrins, can contribute to invasiveness of bladder cancers.General significanceOur findings show that morphological and mechanical stability is directly related to actin filament organization used by cancer cells to adapt to altered laminin-rich microenvironment.     

Actin pedestal formation by enteropathogenic Escherichia coli and intracellular motility of Shigella flexneri are abolished in N-WASP-defective cells

In mammalian cells, actin dynamics is tightly controlled through small GTPases of the Rho family, WASP/Scar proteins and the Arp2/3 complex. We employed Cre/loxP-mediated gene targeting to disrupt the ubiquitously expressed N-WASP in the mouse germline, which led to embryonic lethality. To elucidate the role of N-WASP at the cellular level, we immortalized embryonic fibroblasts and selected various N-WASP-defective cell lines. These fibroblasts showed no apparent morphological alterations and were highly responsive to the induction of filopodia, but failed to support the motility of Shigella flexneri. In addition, enteropathogenic Escherichia coli were incapable of inducing the formation of actin pedestals in N-WASP-defective cells. Our results prove the essential role of this protein for actin cytoskeletal changes induced by these bacterial pathogens in vivo and in addition show for the first time that N-WASP is dispensible for filopodia formation.     

Epithelial–mesenchymal transition in cancer metastasis: Mechanisms,markers and strategies to overcome drug resistance in the clinic

Epithelial–mesenchymal transition (EMT) is a key step during embryogenesis. Accumulating evidence suggests a critical role in cancer progression, through which tissue epithelial cancers invade and metastasise. Cell characteristics are highly affected during EMT, resulting in altered cell–cell and cell–matrix interactions, cell motility and invasiveness. Nevertheless, the demonstration of this process in human cancer has been proven difficult and controversial. Besides the fact that the acquisition of mesenchymal characteristics is not a prerequisite for cell migration/invasion, it is a transient event that concerns only few cells in a tumour mass. The induction of EMT depends on the tumour type and its genetic alterations as well as on its interaction with the extracellular matrix. In parallel, trials for EMT identification in clinical samples lack of a widely accepted methodology, nomenclature and reliable markers. This review summarizes the main EMT characteristics and proposes methodologies for better analysis in vitro. It also highlights recent studies identifying cells with EMT characteristics in human cancer and proposes certain markers to identify them in tumour samples. Finally, it cites the recent literature concerning the mechanisms of drug resistance related to EMT in the context of anti-tumour therapies and proposes related new targets for therapy.     

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.