NF-κB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression (original) (raw)
Upregulation of NF-κB signaling during EMT. EpRas cells represent oncogenic, fully polarized, Ha-Ras–transformed EpH4 mammary epithelial cells that undergo EMT in response to TGF-β both in tumors as well as in collagen gels, giving rise to mesenchyme-like cells (EpRasXT cells) in both cases. EpRasXT cells are characterized by a spindle-like morphology and gain of mesenchymal marker proteins, a phenotype stabilized by an autocrine TGF-β loop in vitro and in vivo (refs. 19, 20; see Figure 1A). We wanted to determine whether NF-κB might play a role in the EMT process. We therefore analyzed whole-cell extracts from exponentially growing EpRas and EpRasXT cells by electrophoretic mobility-shift assay (EMSA). We detected some NF-κB DNA-binding activity in EpRas cells even without stimulation by known inducers of NF-κB and consistently observed a 3- to 4-fold increase in NF-κB DNA-binding activity in EpRasXT cells (Figure 1B).
NF-κB activity is induced during EMT. (A) Schematic illustrates the morphology and epithelial/mesenchymal marker redistribution or expression found in the cell types used in our study. Nontransformed EpH4 mammary epithelial cells were stably transfected with the Ha-Ras oncogene to yield transformed epithelial EpRas cells that undergo EMT upon treatment with TGF-β, resulting in mesenchymal EpRasXT cells further stabilized by an autocrine TGF-β loop. DPP-IV, dipeptidyl peptidase IV; ZO-1, zona occludens 1. (B) EMSAs of whole-cell extracts (6 μg) of exponentially growing EpRas and EpRasXT cells were performed with an NF-κB–specific probe (upper panel) and with an octamer-specific probe (Oct; lower panel) used as a control. Quantified relative DNA-binding levels are indicated below the EMSAs. Similar data were obtained using different protein extract preparations (see also Figure 2A).
Based on those observations, we next asked whether NF-κB target genes were induced in mesenchymal EpRasXT cells to the same degree as their epithelial counterpart, EpRas cells. To address this question, we reanalyzed the data from a previously reported expression profile, in which we had performed microarray analysis of polysome-bound mRNAs to identify genes differentially expressed in EpRasXT compared with EpRas cells (Tables 2 and 3 in ref. 18). Interestingly, 13 of the 75 annotated genes upregulated during EMT had previously been described as NF-κB target genes (Table 1). In mesenchymal EpRasXT cells, several genes encoding NF-κB–regulated cytokines/chemokines (IL-11, JE/MCP-1, and KC/Gro1), proteases (MMP-13, cathepsin B, MMP-2, and cathepsin Z), hormones (cholecystokinin and placental proliferin 2), and the transcription factor Stat-1, as well as β_2-microglobulin_ were expressed at elevated levels compared with their epithelial counterparts. Furthermore, NF-κB has previously been shown to directly regulate the two EMT marker genes vimentin and tenascin C (Table 1). In addition, bcl-3, a regulator of NF-κB activity (24) and previously identified as being expressed in human breast cancer (25), is likewise induced in EpRasXT cells (18). In contrast, among the genes downregulated in EpRasXT cells, only one gene (thrombospondin 1) has been suggested to be regulated by NF-κB (ref. 26; reviewed in ref. 27). Thus, expression profiling results clearly indicate an enriched expression of NF-κB target genes in mesenchymal EpRasXT cells, consistent with increased activity of NF-κB.
NF-κB–regulated genes induced during EMT in the EpRas/EpRasXT cell pair
TGF-β induces NF-κB activity during EMT. Based on the observation that TGF-β signaling is essential for the induction of EMT in EpRas cells (19, 20), as well as reports that TGF-β can modulate NF-κB activity in certain epithelial cells (28), we sought to test whether TGF-β can affect NF-κB activity in EpRas and EpRasXT cells. In order to characterize changes in NF-κB DNA-binding activity after stimulation with TGF-β1, we incubated cultures of EpRas and EpRasXT cells in the presence of TGF-β1 and monitored the levels of NF-κB DNA-binding activity by EMSA. In EpRas cells, we observed a 3- to 4-fold induction of NF-κB DNA-binding activity within 30 minutes, while we observed no response to TGF-β in EpRasXT cells (Figure 2A). The result with the EpRasXT cells was not surprising, given that the cells themselves produce TGF-β. In addition, EpRas and EpRasXT cells were transiently transfected with an NF-κB–dependent luciferase reporter (3xκB.luc). Stimulation of EpRas cells with TGF-β1 resulted in an increase of roughly 2-fold in NF-κB transactivation activity within 2–8 hours (Figure 2B), whereas no increase was noted in EpRasXT cells (data not shown). Thus, TGF-β stimulation leads to an induction of functionally active NF-κB in EpRas cells, while it does not affect the increased NF-κB activity in EpRasXT cells.
TGF-β induces NF-κB activity in EpRas cells. (A) EpRas and EpRasXT cells were stimulated with TGF-β1 (5 ng/ml) for the indicated times. EMSA with whole-cell extracts (6 μg) was performed with an NF-κB–specific probe (upper panel) and with an octamer-specific probe (lower panel) used as a control. (B) NF-κB transcriptional activity. EpRas cells were transiently transfected in triplicate with a 3xκB.luc or β-globin-TATA reporter construct. At 20 hours after transfection, cells were treated with TGF-β1 (5 ng/ml) for the indicated times. Then, the luciferase activities of extracts were determined and were normalized based on Renilla luciferase expression. The ratio of 3xκB.luc and β-globin-TATA is shown. The expression level of unstimulated empty vector-infected EpRas cells was used as the reference luciferase activity and was arbitrarily set to 1. Means and standard deviations are representative of two independent experiments carried out in triplicate. Bars represent standard deviations.
NF-κB is essential for EMT. To study the contribution of the IKK/IκBα/NF-κB signaling module in the regulation of EMT and metastasis, we used retroviral gene transfer to express dominant interfering mutants of this pathway in EpRas cells. Infections were performed using retroviruses expressing a _trans_-dominant (TD) IκBα protein (TD-IκBα, in which serine residues at positions 32 and 36 are mutated to alanine residues, resulting in a nondegradable repressor), a constitutively active (CA) IKK-2 protein (CA–IKK-2, in which two serine residues in the activation loop are mutated to phosphomimetic glutamic acid residues), or an empty vector control. Stably infected cells were visualized by immunofluorescence microscopy, as the retroviruses coexpress enhanced GFP. Expression of TD-IκBα and CA–IKK-2 in cells from the φNX producer line and in stably infected EpRas cells was assessed by Western blot (Figure 3, A and B). This analysis revealed strong overexpression of the mutant proteins compared with that of the endogenous counterparts. As previously observed in another cellular system (29), the presence of high levels of exogenous TD-IκBα resulted in reduced expression of endogenous IκBα, most likely due to inhibition of NF-κB activity. The effects of TD-IκBα and CA–IKK-2 expression in EpRas cells on NF-κB DNA-binding activity were analyzed by EMSA. In EpRas cells infected with TD-IκBα, no NF-κB DNA-binding activity was observed, regardless of whether cells were left unstimulated or were stimulated with TGF-β (Figure 3C), TNF-α, or PMA (data not shown). In contrast, cells infected with CA–IKK-2 exhibited 2- to 3-fold increased DNA-binding activity in the unstimulated state (compared with unstimulated EpRas control cells), and showed a roughly 2-fold higher induction of NF-κB activity after being stimulated with TGF-β, compared with TGF-β–treated EpRas control cells. Transient transfection of these cells with 3xκB.luc and subsequent luciferase assays revealed a 3- to 4-fold induction of luciferase activity in untreated EpRas cells infected with CA–IKK-2 and a more than 2-fold higher induction upon treatment with TGF-β, compared with that of unstimulated or TGF-β–treated EpRas control cells, respectively. In contrast, NF-κB transcriptional activity was virtually completely inhibited before and after treatment with TGF-β in EpRas cells expressing TD-IκBα (Figure 3D). In addition, RT-PCR analysis of a subset of NF-κB–regulated target genes that are associated with EMT (Table 1) (18) was performed with the EpRas mutants before and after TGF-β–induced EMT. This analysis showed induction of MMP-13, MCP-1, and cholecystokinin in CA–IKK-2–expressing EpRas cells in the absence of TGF-β (in the case of MCP-1, this was even comparable to the expression level in EpRasXT cells), and slightly stronger expression of these genes in the presence of TGF-β (cholecystokinin and MMP-13), whereas expression and TGF-β–induced upregulation of these genes were almost completely blocked in TD-IκBα–expressing cells (Figure 3E). Based on these results, NF-κB activates at least a subset of genes in the TGF-β–induced genetic program underlying EMT.
Modulation of NF-κB activity in EpRas cells. (A and B) Expression levels of dominant interfering mutants in ΦNX producer cells (A) and in stably infected EpRas cells (B) were determined by Western blot analysis of whole-cell extracts, using IκBα- and IKK-specific antibodies CA–IKK-2, endogenous IKK-2 (comigrating with CA–IKK-2), TD-IκBα, and endogenous IκBα. Equal loading was assessed using a p65/RelA–specific antibody (B, bottom panel). (C) Extracts from stably infected EpRas mutants left untreated (–) or stimulated with TGF-β (5 ng/ml) for 2 hours (+) were analyzed by EMSA with an NF-κB–specific probe (upper panel) and with an octamer-specific probe (lower panel) as loading control. Quantified relative DNA-binding levels are indicated. (D) Stably infected cells were transiently transfected in triplicate with 3xκB.luc or β-globin-TATA reporter constructs. After 24 hours, cells were treated with TGF-β (5 ng/ml for 4 hours) (+) or were left untreated (–). Luciferase activity was measured and normalized based on Renilla luciferase expression (as described in Figure 2B). The ratio of 3xκB.luc and β-globin-TATA is shown. Expression levels of unstimulated empty vector–transfected EpRas cells were used as a reference and arbitrarily set to 1. Bars represent standard deviations. (E) EpRas mutants and EpRasXT cells were stimulated with TGF-β (5 ng/ml) for the times indicated, and RT-PCR analysis was carried out for transcript expression of MMP-13, MCP-1, cholecystokinin, and β_-actin_, as described in Methods. C, control (water); Empty, EpRas cells infected with empty vector control.
We then asked whether this modulation in NF-κB activity affected the ability of EpRas cells to undergo EMT. On porous support (filters) allowing epithelial polarization, EpRas cells infected with empty vector (like uninfected cells) showed a fully polarized epithelial phenotype with basolateral plasma membrane expression of the epithelial marker E-cadherin, but no expression of the mesenchymal marker vimentin (Figure 4, A–D). Treatment of these EpRas cells with TGF-β for 5 days resulted in strands of spindle-shaped, vimentin-positive cells only weakly expressing E-cadherin (Figure 4, A, B, and D). No phenotypic changes or changes in marker expression compared with EpRas control cells were observed in TD-IκBα–expressing cells in the absence of TGF-β (Figure 4, A–D). Upon treatment with TGF-β, however, a considerable proportion of these TD-IκBα–overexpressing EpRas cells with blocked NF-κB activity rapidly detached from porous supports as a consequence of cell death. The remaining cells almost completely failed to undergo EMT (Figure 4A). TGF-β–treated TD-IκBα–expressing EpRas cells lacked the strands of spindle-shaped mesenchymal cells that were abundant in empty virus–infected EpRas control cultures (Figure 4, A and B). The same cells failed to upregulate vimentin and retained high levels of E-cadherin, which was partially redistributed to the cytoplasm, indicating some loss of polarity (Figure 4D). Surprisingly, CA–IKK-2–overexpressing EpRas cells with increased NF-κB activity were able to undergo EMT at a considerable rate even in the absence of TGF-β (Figure 4, A and B). After cells had grown for 6 days on porous support, we observed strands of spindle-shaped, E-cadherin–negative and vimentin-positive cells that covered more than 10% of the total surface area (Figure 4, A, B, and D). When we analyzed bulk cultures of these cells by Western immunoblot, we observed a strong reduction in E-cadherin levels (Figure 4C). Consistent with these observations, we noted enhanced EMT in CA–IKK-2–expressing cells upon TGF-β treatment compared with that of control EpRas cells, as indicated by a higher percentage of spindle-shaped cells with cytoplasmic (depolarized) or no E-cadherin expression, and strong vimentin expression (Figure 4, A, B, and D). Importantly, very similar results were obtained when the CA-IKK-2 and TD-IκBα transgenes were introduced into V12S35Ras cells. These cells represent another monogenic transformed cell line, independently generated from the original EpH4 cells and transformed by an effector mutant of a different oncogenic Ras that induces hyperactive ERK/MAPK but not PI3K signaling (17). We observed an inhibition of EMT in TD-IκBα–expressing cells and a considerable rate of spontaneous EMT in the absence of TGF-β in CA–IKK-2–expressing V12S35Ras cells that occurred even more frequently than in CA–IKK-2–expressing EpRas cells (see Supplemental Figure 1; supplemental material available at http://www.jci.org/cgi/content/full/114/4/569/DC1). Thus, the role of NF-κB in the regulation of EMT is not limited to a single cell line.
EpRas cells expressing TD-IκBα fail to undergo EMT, while CA–IKK-2 expression drives EMT in the absence of TGF-β (analysis on porous support). EpRas cells expressing the empty vector control, TD-IκBα, or CA–IKK-2 were cultivated on porous supports for 7 days in the presence (+) or absence (–) of TGF-β (5 ng/ml; days 2–7). (A) Photographs of cultivated cells are shown; regions with strands of spindle-shaped mesenchymal cells are indicated by red dotted lines. Original magnification, ×100. (B) Quantification of the area on porous supports covered by mesenchymal strands as percentage relative to total area covered by adherent cells (cultures shown in A; day 6). (C) E-cadherin levels in EpRas cells expressing empty vector, TD-IκBα, or CA–IKK-2 and not treated with TGF-β were determined by Western blot analysis with an E-cadherin–specific antibody (E-Cad; upper panel). Subsequent stripping and reprobing of the blot with a p65/RelA–specific antibody (lower panel) was done to demonstrate equal loading. (D) Cells as indicated were cultivated on porous supports for 7 days in the presence or absence of TGF-β (5 ng/ml; days 2–7). Cells were immunostained for E-cadherin or vimentin (red) with DAPI counterstaining for DNA (blue), as described in Methods. Regions of mesenchymal, E-cadherin–negative and vimentin-positive cells are outlined by white dotted lines and correspond to the “mesenchymal” areas shown in A (red dotted lines). Original magnification, ×400.
A more physiological culture system to analyze epithelial cell behavior and plasticity are three-dimensional serum-free collagen I gel cultures (15). In these cultures, fully polarized EpRas control cells form tubular and alveolar structures with large lumina (Figure 5A). These cells show basolateral membrane (polarized) expression of E-cadherin, but no vimentin expression (Figure 5C). Upon addition of TGF-β1, EpRas control cells underwent EMT, as shown by a spindle-shaped and migratory phenotype, loss of E-cadherin, and de novo expression of vimentin after 6 days of treatment (Figure 5, A and C). Untreated TD-IκBα cells resembled control EpRas cells in that they formed epithelial structures (tubular structures with lumina) and showed basolateral E-cadherin staining and no vimentin expression. Despite this resemblance, TD-IκBα epithelial structures appeared more compact and smaller in size (Figure 5, A and C). Upon treatment with TGF-β, epithelial structures formed by TD-IκBα–expressing EpRas cells retained E-cadherin expression (Figure 5C), but rapidly disintegrated (Figure 5, A and C). Only a very small fraction of the structures (about 0.5%) formed unordered cell strands before disintegration (data not shown). In contrast, untreated CA–IKK-2–expressing cells with increased NF-κB activity formed two types of structures. While a large proportion of epithelial tubular structures with lumina were apparent (Figure 5A), a significant number of the structures consisted of unordered cell strands with spindle-like cellular morphology, resembling control EpRas cells treated with TGF-β (Figure 5, A and B). Interestingly, among the epithelial structures resembling control EpRas cells (E-cadherin positive and vimentin negative), a large percentage had either lost or downregulated expression of CA–IKK-2, as indicated by low levels of the coregulated GFP expression (Figure 5C, bottom). In contrast, mesenchymal structures generated by untreated CA–IKK-2–expressing cells were E-cadherin negative, expressed vimentin, and showed strong GFP staining, indicative of high transgene expression (Figure 5C). Thus, high levels of CA–IKK-2 expression appear to promote EMT even in the absence of TGF-β. As expected, TGF-β treatment induced the remaining epithelial CA–IKK-2–expressing EpRas cells to rapidly form mesenchymal structures expressing vimentin but no E-cadherin (Figure 5, A and C). We also determined whether a strong NF-κB activator such as TNF-α would induce EMT. Indeed, we observed a small number of mesenchymal structures expressing vimentin in TNF-α–treated EpRas collagen gel cultures, even in the absence of TGF-β (Supplemental Figure 2). In conclusion, an activated NF-κB pathway plus Ras is sufficient to cause EMT, whereas inhibition of NF-κB activity prevents EMT, causing disintegration of structures formed in collagen.
TD-IκBα expressed in EpRas cells prevents EMT, whereas CA–IKK-2 induces EMT in the absence of TGF-β (analysis in collagen gels). EpRas cells expressing the empty vector control, TD-IκBα, or CA–IKK-2 were seeded into collagen gels, were allowed to form structures for 3–5 days, and were left untreated for no induction (–) or were induced to undergo EMT by the addition of TGF-β (+) for 5–6 days. (A) Left column, culture without TGF-β for 7 days; middle column, culture without TGF-β for 5 days, plus TGF-β treatment (5 ng/ml) for 1 day; right column, culture without TGF-β for 5 days, plus TGF-β treatment (5 ng/ml) for 5 days. Photographs of representative tubular structures with lumina (yellow arrows) or distended chords and strands of invasive cells with mesenchymal morphology (white arrows) are shown. Original magnification, ×100. (B) Quantification of mesenchymal structures (see white arrows in A for the CA–IKK-2, –TGF-β culture) as the percentage relative to that of 60 randomly chosen structures per gel after 3 days of culture in the absence of TGF-β. Below graph, n indicates the number of collagen gels analyzed. Bars represent the standard deviations obtained in analyzing individual collagen gels. (C) Collagen gel structures were stained for the epithelial marker E-cadherin (first and second columns) or the mesenchymal marker vimentin (third and fourth columns). Bottom row, second from left (CA–IKK-2–expressing cells, –TGF-β): GFP expression (green) of structures stained for E-cadherin. Inset, third panel from left, middle row (TD-IκBα–expressing cells, +TGF-β): disintegrating structure stained for E-cadherin. Inset, second panel from right, bottom row (CA–IKK-2–expressing cells, –TGF-β): GFP expression (green) of structures stained for vimentin. Original magnification, ×400.
NF-κB is required both for protection from TGF-β–induced apoptosis and for direct promotion of EMT. The previous experiments showed that NF-κB can, at least in part, substitute for TGF-β in the induction of EMT in collaboration with Ras. We next addressed the mechanistic consequence of NF-κB inhibition with respect to the prevention of EMT. Because we observed rapid disintegration of TD-IκBα–expressing structures upon TGF-β treatment, we assessed their apoptotic response under these conditions. EpRas control cells as well as TD-IκBα– and CA–IKK-2–expressing derivatives were allowed to form organotypic structures in collagen gels for 3 days. Then they were induced to undergo EMT by TGF-β or were left untreated. TGF-β–treated TD-IκBα–expressing EpRas structures showed cell disintegration due to apoptosis, as determined by in situ TUNEL staining (Figure 6A). As shown in Figure 6B, approximately 55% of these cells were TUNEL positive and were thus apoptotic after 6 days of TGF-β treatment. Moreover, TD-IκBα cells showed a slight elevation in induction of apoptosis compared with control EpRas cells even in the absence of TGF-β (4.5% versus 0.6%), possibly explaining the smaller size of epithelial structures formed by TD-IκBα–expressing cells in collagen gels. Finally, EpRas cells expressing CA–IKK-2 showed low levels of apoptosis, comparable to that of EpRas control cells. We then asked whether the observed failure of TD-IκBα–expressing EpRas cells to undergo EMT (Figures 4 and 5) was exclusively due to inhibition of the antiapoptotic function of NF-κB in these cells. EpRas control cells and TD-IκBα–expressing derivatives were cultured on porous support. Treatment of TD-IκBα–expressing EpRas cells with 25 μM cell-permeable caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) strongly suppressed the TGF-β–induced apoptosis seen in these cells (on porous support as well as in collagen gels; Figure 6B), to a level that was comparable to that of EpRas control cells (data not shown). Importantly, neither mesenchymal structures (as seen in TGF-β–treated EpRas control cells) nor loss of polarized E-cadherin expression or de novo vimentin expression was observed in these Z-VAD-FMK–treated TD-IκBα–expressing EpRas cells after 4 days of stimulation with TGF-β (Figure 6, C and D). Notably, due to the significant fraction of apoptotic cells following TGF-β treatment, small areas of irregular structures were detected in the TD-IκBα–expressing EpRas cultures (data not shown). These were not observed when apoptosis was blocked by Z-VAD-FMK. In summary, NF-κB signaling is required in Ras-transformed cells for protection from TGF-β–induced apoptosis during EMT. Moreover, NF-κB plays an additional role as a direct regulator of the EMT program, as blockade of apoptosis does not restore the ability of TD-IκΒα–expressing EpRas cells to undergo EMT in response to TGF-β.
Suppression of NF-κB activity in EpRas cells leads to apoptosis and prevents EMT. (A) EpRas cells expressing the empty vector control, TD-IκBα, or CA–IKK-2 were seeded into collagen gels, were allowed to form structures for 3 days, and were treated with TGF-β for 6 days or were left untreated. Collagen cultures were subjected to in situ TUNEL staining (red) and DAPI staining (blue, indicating living cells). Light microscopy images of collagen gels from the same experiment photographed prior to TUNEL staining are also shown. Arrows indicate TUNEL-positive nuclei. Inset, right middle panel: another structure with TUNEL-positive nuclei. Original magnification, ×200. (B) Quantification of TUNEL-positive cells from collagen gel structures shown in A. TUNEL staining of nuclei was assessed in at least 300 cells from three to six randomly chosen fields. The average from two to three collagen gels was used to calculate the apoptotic index and standard deviation. Average percentage of apoptotic cells, assessed in 2–3 collagen gels (at least 300 cells per gel) for each cell type, are indicated above bars. (C) EpRas cells expressing the empty vector control or TD-IκBα were cultivated for 5 days on porous support in the presence or absence of 25 μM Z-VAD-FMK (Z-VAD; days 0–5) and/or TGF-β (5 ng/ml; days 1–5). The quantification of the area on porous support covered by mesenchymal strands is shown as the percentage relative to the total area covered by adherent cells (at day 4, after 3 days with or without TGF-β treatment). (D) Cells as indicated (with or without 25 μM Z-VAD-FMK; days 0–5) were cultivated on porous support for 5 days in the presence or absence of TGF-β (5 ng/ml; days 1–5). Cells were immunostained for E-cadherin or vimentin (red) plus DAPI counterstaining for DNA (blue) after 5 days of culture on porous support. Original magnification, ×400.
Inhibition of NF-κB activity in mesenchymal EpRasXT cells causes reversal of EMT. We next addressed whether interference with NF-κB activity would also affect the mesenchymal EpRasXT cells that have completed EMT. EpRasXT cells were again stably infected with retroviruses expressing TD-IκBα or CA–IKK-2 or with a GFP-only empty control vector. Transgene expression, as assessed by Western blot analysis, is shown in Figure 7A. EMSA showed complete inhibition of NF-κB DNA-binding activity in untreated and TNF-α- or PMA-stimulated EpRasXT cells expressing TD-IκBα. The expression of CA–IKK-2 resulted only in a moderate (less than 2-fold) enhancement of NF-κB activity in untreated cells (Figure 7B). Transient transfection with 3xκB.luc and subsequent luciferase assays revealed a strong blockade of NF-κB transactivation activity in EpRasXT cells expressing TD-IκBα, while a moderate increase of luciferase activity was observed in EpRasXT cells infected with CA–IKK-2.
Effect of retrovirus-expressed TD-IκBα and CA–IKK-2 on NF-κB activity in EpRasXT cells. (A) Expression levels of dominant interfering mutants compared with that of their endogenously expressed wild-type counterparts in stably infected EpRasXT cells were determined by Western blot analysis of whole-cell extracts using IκBα- and IKK-specific antibodies for visualization. Protein bands of CA–IKK-2/endogenous IKK-2 (comigrating), TD-IκBα, and endogenous IκBα are indicated. Equal loading was assessed by stripping and reprobing of blots with a p65/RelA antibody (bottom panel). (B) Stably infected EpRasXT cells were not treated (control) or were stimulated with TNF-α (TNF; 40 ng/ml) for 1 hour or with PMA (50 ng/ml) for 1 hour. Then, NF-κB DNA-binding activity was assessed with an NF-κB–specific probe (upper panel) or with an Sp-1–specific probe (lower panel) as a loading control. (C) NF-κB transcriptional activity in the EpRasXT derivatives described in A and B was determined by luciferase assay, as described in Figure 3D. The expression level of empty vector–transfected EpRasXT cells was used as a reference luciferase activity and was arbitrarily set to 1. Mean values are indicated above bars.
To test whether TD-ΙκBα was able to revert EMT, we cultured EpRasXT cells expressing TD-IκBα, CA–IKK-2, or the control vector on porous support. As expected, CA–IKK-2– and control vector–expressing EpRasXT cells showed a mesenchymal, spindle-shaped phenotype and expressed high levels of vimentin, but no E-cadherin (Figure 8, A and B). Interestingly, however, a large percentage of TD-IκBα–expressing cells reverted to an epithelial phenotype, in which the cells formed compact structures, regained marked E-cadherin expression at the plasma membrane, and almost completely lost expression of vimentin, as demonstrated by immunofluorescence (Figure 8A) and Western blot analysis (Figure 8B). Similar results were obtained under different culture conditions (data not shown). TD-ΙκBα–expressing EpRasXT cells showed no obvious signs of cell death when cultured on porous support (e.g., condensed nuclei, disintegrated cells, and detachment from porous support). Annexin V staining (Figure 8C) as well as cell cycle analysis (data not shown) of TD-IκBα–expressing EpRasXT cells that had reverted to an epithelial phenotype demonstrated that reverted epithelial cells were still viable and healthy. These results indicate that NF-κB activity is required for maintenance of the mesenchymal phenotype of Ras-transformed cells that have undergone EMT.
Inhibition of NF-κB activity in mesenchymal EpRasXT cells causes reversal of EMT. EpRasXT cells expressing the empty vector control, TD-IκBα, or CA–IKK-2 were cultivated on porous supports for 6 days. (A) Cells were immunostained for E-cadherin (red; left column) or vimentin (red; right column) and were counterstained for DNA (blue; omitted in top and middle panels, right column). Original magnification, ×200. (B) E-cadherin and vimentin levels were determined by Western blot analysis with antibodies specific for E-cadherin and vimentin, respectively. Subsequent stripping and reprobing of the blot with a p65/RelA–specific antibody was carried out to show equal loading (bottom panel). (C) EpRasXT cells infected with either the TD-IκBα–expressing virus or the control virus were grown on porous supports to allow EMT reversal in TD-IκBα cells. Apoptotic cells were identified by annexin V staining and FACS analysis.
NF-κB is required for the metastatic potential of EpRas cells in vivo. EpRas cells undergo EMT in vivo in response to endogenous TGF-β (19). TGF-β–induced EMT of EpRas cells is tightly linked to their ability to form lung metastases, evident after tail vein injection of cultured EpRas cells (S. Grünert and H. Beug, unpublished data) or cells recultivated from EpRas-induced primary mammary tumors (17). Because NF-κB activity was found to be essential for both the induction and maintenance of EMT, we sought to determine whether NF-κB signaling is also required for metastatic potential induced by Ras plus TGF-β in vivo and whether inhibition of NF-κB would abrogate this metastatic ability. The metastatic potential of TD-IκBα–expressing EpRas cells and EpRas control cells was assayed by injection of cultured cells into the tail vein of nude mice. Mice injected with EpRas control cells rapidly died from numerous large metastases (on average, 3–4 weeks after tail vein injection), while mice receiving TD-IκBα–expressing EpRas cells appeared healthy at the time of death of the mice injected with EpRas control cells. Mice injected with EpRas cells with blocked NF-κB activity showed a 2-fold decrease in lung weight (compared with that of control mice of similar age; data not shown) and had only a few small (micro-) metastases (average number, 16.6 metastases per lung) by histological analysis, compared with an average number of 171.0 metastases per lung in animals injected with EpRas control cells (Figure 9, A–C). To verify that TD-IκBα–expressing EpRas cells were still able to form primary tumors in a fashion similar to that of EpRas control cells, we injected the cell types described above (in each case from the same batches as used for tail vein injections) into mammary gland fat pads of nude mice. After 3 weeks, both EpRas control cells as well as EpRas cells expressing TD-IκBα formed tumors, which differed mainly in size (Figure 9D). In conclusion, inhibition of NF-κB activity strongly affects the metastatic potential of EpRas cells in vivo, while primary tumor formation is affected only moderately.
Inhibition of NF-κB activity prevents metastasis of EpRas cells. (A) EpRas cells infected with empty GFP vector (Empty) or TD-IκBα were injected (5 × 105 cells per injection) into the tail veins of nude mice (four animals per cell type), and mice were analyzed for the presence of lung metastases. Note large metastatic nodules (white arrows) in the lung from the mouse injected with EpRas cells that had been infected with empty vector (EpRas empty; left). (B) Metastases in lungs similar to those shown in A were quantified in serial sections to determine the mean numbers of metastases per lung (four lungs per cell type). (C) Hematoxylin and eosin staining of lungs from mice injected with EpRas cells that had been infected with empty vector or TD-IκBα. Note large metastases in the mouse injected with EpRas cells that had been infected with empty vector. Original magnification, ×10. (D) The same cells analyzed in A–C were injected (2 × 105 cells per injection site; four injection sites per animal) into the fat pads of nude mice (three animals per cell type), and total tumor weight was determined for each individual mouse after 3 weeks. Average total tumor weights are shown.