EMT and scattering: two distinct processes in epithelial plasticity induced by growth factors or oncogenes
In addition to oncogenic Ras (Oft et al., 1996
), hepatocyte growth factor (HGF)/scatter factor (SF), fibroblast growth factor (FGF), and TGFβ alone induce mesenchymal features in diverse epithelial cell systems (Brinkmann et al., 1995
; Piek et al., 1999
; Thiery and Chopin, 1999
). We investigated whether these different signals would induce similar or different types of mesenchymal phenotypes in EpRas cells. These represent Ha-Ras–transformed fully polarized EpH4 cells (Oft et al., 1996
), spontaneously immortalized from mammary glands of midpregnant mice (Reichmann et al., 1992
). EpRas cells were seeded in serum-free collagen gels and subjected to different factor treatments. HGF/SF induced EpRas cells to invade collagen gels and grow as disordered chords of nonpolarized cells ( A; true for >95% of >100 structures counted in two to three separate collagen gels; see Materials and methods). Upon HGF/SF removal, >90% of these factor-induced disordered structures reverted to hollow tubular structures after an additional 5 d ( B). The disordered collagen gel structures formed by the HGF/SF-treated cells continued to express β4-integrin, E-cadherin, and ZO-1 at the entire cell surface (indicating loss of polarity) and failed to express vimentin ( C, left; unpublished data). Upon factor withdrawal, β4-integrin regained polarized basal expression in the hollow tubules formed ( C, right). Likewise, E-cadherin and ZO-1 were again expressed basolaterally ( C, inset; unpublished data). Similar results were obtained using FGF (unpublished data).
Figure 1. Scattering induced by scatter factor (HGF/SF) and TGFβ can be distinguished from EMT by reversibility and lack of marker changes. (A–D) EpRas collagen gel structures were treated with SF/HGF and TGFβ. (A) EpRas cells seeded in (more ...)
Controls using EpRas cells undergoing EMT in response to TGFβ showed that the spindle-like phenotype induced by TGFβ treatment ( A, inset) persisted after factor removal ( B, inset) and involved loss of E-cadherin/β4-integrin, whereas vimentin was strongly induced ( D, left). Again, these marker changes persisted after TGFβ removal ( D, right). These results show that HGF and FGF reversibly induce a spindle-like migratory phenotype in EpRas cells, which is characterized by loss of polarity but involves no lasting changes in epithelial or mesenchymal marker expression. We refer to this phenotype as scattering, since FGF and HGF also induce “classical” scattering on plastic (unpublished data), and distinguish it from EMT, which is not reversed upon factor removal and involves stable loss of epithelial markers and upregulation of mesenchymal markers.
To answer the question if and how TGFβ alone (Piek et al., 1999
) would alter the phenotype of nontransformed EpH4 cells, we used EpH4 cells expressing a retrovirally transduced antiapoptotic Bcl-2 protein. This was necessary since doses of TGFβ (2–5 ng/ml) causing EMT in EpRas cells induced growth arrest and apoptosis in EpH4 cells. In the absence of TGFβ, the Bcl-2–expressing EpH4 cells formed tubular structures in collagen gels similar to EpH4 control cells ( compared with F). When treated with TGFβ, the EpH4–Bcl-2 cells were apoptosis protected as expected (8% TUNEL-positive cells; G, inset) and formed disordered structures consisting of migratory cells ( G), whereas control EpH4 cells underwent apoptosis (48% TUNEL-positive cells; E, inset). However, in contrast to EpRas cells the TGFβ-treated Bcl-2–expressing EpH4 cells reverted to hollow tubular structures 4 d after TGFβ removal ( H). Likewise, they maintained E-cadherin expression throughout the experiment, whereas vimentin expression was not detectable (unpublished data). In conclusion, TGFβ causes reversible scattering in EpH4 cells apoptosis protected by Bcl-2 but fails to induce EMT in the absence of oncogenic Ras, suggesting that Ras induces an EMT competent state in addition to prevent TGFβ-induced apoptosis.
Active oncogenic Ras is required for both induction and maintenance of EMT
Using a kinase-dead dominant negative mutant of the TGFβ-RII, we showed previously that maintenance of TGFβ-R signaling is required for EMT and metastasis (Oft et al., 1998
). To address if EMT also required maintenance of oncogenic Ras activity, we used a specific nontoxic inhibitor of Ras-farnesylation (L739749 [Kohl et al., 1994
]). L739749 reversed EMT in mesenchymal EpXT cells, causing reformation of hollow tubular structures ( B) from the unordered cords and strands of EpXT cells ( A) but did not affect EpH4 ( B, inset). After inhibitor withdrawal, the reverted cells maintained their polarized phenotype as indicated by persistence of tubular structures ( C). However, these structures could be reinduced to undergo EMT when again treated with TGFβ (unpublished data). This indicates that Ras inhibition reversed EMT completely, converting EpXT cells into cells with EpH4 cell properties. In line with this, reactivation of Ras in the reverted cells was insufficient for EMT, again requiring TGFβ as in the original EpRas cells (Oft et al., 1996
). Furthermore, cotreatment of the reverted cells with L739749 and TGFβ resulted in cell death ( D), whereas their treatment with Ras inhibitor alone caused no phenotypical changes ( D, inset).
Figure 2. Ras activity is required for induction and maintenance of EMT. (A–D) EpXT cells were seeded into collagen gels in the absence (A) or presence (B) of 10 μM Ras farnesylation inhibitor (L739749), and resulting structures were photographed (more ...)
These results were confirmed by analyzing marker expression, using ultrathin cryosections stained with respective fluorescent antibodies (see Materials and methods) from similar collagen gel structures as shown in . As expected, the mesenchymal chords of untreated EpXT cells expressed vimentin and intracellular TGFβ ( E, top) and deposited fibronectin extracellularly (periphery of structures and intercellular spaces; E, bottom). After treatment with L739749 for 5 d ( F), vimentin and TGFβ expression was no longer detectable in the hollow structures obtained ( F, top). Two additional mesenchymal markers, CD68 (macrosialin) and calgranulin A, were also downregulated (unpublished data). Furthermore, the L739749-treated cells reexpressed the tight junction marker ZO-1 ( F, bottom) and E-cadherin (unpublished data) in a polarized basolateral fashion. The presence of tight junctions in the epithelial tubules induced by L739749 were confirmed by EM, using immunogold staining for ZO-1, although the mesenchymal chords obtained in the absence of inhibitor failed to show any junction-like structures (unpublished data).
In conclusion, inhibition of Ras in EpXT cells completely restores a normal polarized epithelial phenotype, indicating that sustained oncogenic Ras and TGFβ-R signaling are required for induction and maintenance of EMT in EpRas cells.
Ras downstream signaling required for EMT: analysis by low molecular weight inhibitors
Oncogenic Ha-Ras activates multiple downstream signal transduction pathways, including the Raf/MAPK module and the PI3K-PKB/Akt pathway (for review see Rommel and Hafen, 1998
). Both pathways were implied in cell transformation in vitro (Rodriguez-Viciana et al., 1997
) and tumorigenesis (Webb et al., 1998
). To dissect Ras-activated downstream pathways required for different aspects of Ha-Ras transformation in epithelial cells (EMT, scattering, and apoptosis protection), we employed two specific low molecular weight inhibitors selectively blocking either the MAPK pathway (PD98059) or PI3K signaling (LY294002).
The activity of the Mek1/MAPK- and PI3K-PKB/Akt pathways in EpRas and EpH4 cells and the ability of the two inhibitors to selectively suppress either pathway was analyzed by Western blots using phospho-specific antibodies to MAPK/extracellular signal–regulated kinase (Erk)1/2 and PKB/Akt. EpRas cells exhibited strongly elevated phospho-Erk/MAPK and phospho-PKB/Akt levels compared with EpH4 cells ( A, lanes E and R). These elevated levels were largely independent of cell density (unpublished data). Testing of the inhibitors in EpRas cells revealed that 10 μM PD98059 reduced MAPK phosphorylation to basal levels, whereas 40 μM completely abolished MAPK phosphorylation (unpublished data). Conversely, PKB/Akt phosphorylation levels were not affected by PD98059 ( A, left, lanes R and iR; unpublished data). Conversely, 5 μM LY294002 added at 12-h intervals stably reduced elevated PKB/Akt phosphorylation in EpRas cells to basal levels ( A, middle; unpublished data), whereas 30 μM LY294002 essentially abolished PKB/Akt phosphorylation ( A, right). In contrast, even high levels of LY294002 had no effect on MAPK phosphorylation in EpRas cells ( A).
Figure 3. Inhibitors of Mek-1 prevent and reverse EMT, and PI3K inhibitors abolish protection from TGFβ-induced apoptosis. (A) EpH4 cells (lanes marked E) or EpRas cells mock treated (lanes marked R) or treated with the inhibitors indicated (lanes marked (more ...)
Next, we analyzed the effects of the two inhibitors on TGFβ-induced EMT in collagen gels ( B, top). 10 μM PD98059 prevented EMT in >95% of the structures, giving rise to hollow tubules ( B, top) typical for control EpRas cells ( A, inset). These inhibitor-treated structures failed to express vimentin while retaining E-cadherin (unpublished data). In contrast, 5 μM LY294002 did not affect TGFβ-induced EMT in EpRas cells ( B, bottom left; unpublished data), whereas 30 μM LY294002 (almost completely suppressing PI3K signaling; A) caused cell disintegration ( B, bottom right). None of the inhibitors strongly altered formation of hollow structures in the absence of TGFβ, showing that they were nontoxic at the respective concentrations used (, A–D, insets).
The disintegration of EpRas cells observed after treatment with 30 μM LY294002 plus TGFβ was clearly due to strongly increased apoptosis as shown by TUNEL staining or dye exclusion (; see Materials and methods). In contrast, 10 μM PD98059 or 5 μM LY294002 plus TGFβ and LY294004, PD98059, or TGFβ added alone failed to significantly increase apoptosis (; unpublished data). In conclusion, high levels of Mek1/MAPK activity are necessary for induction and maintenance of EMT. Similar elevated levels of PI3K pathway activity are required for protection from TGFβ-induced apoptosis but not for EMT.
Effector-specific Ras mutants in vitro: MAPK hyperactivation is required for EMT, whereas PI3K signaling causes scattering
To verify these results by an independent approach, two well-characterized Ras mutants were used, which selectively signal along the MAPK pathway, S35-V12–Ras (S35-Ras), or the PI3K pathway, C40-V12–Ras (C40-Ras), due to specific amino acid changes in the effector loop of the Ras protein (Rodriguez-Viciana et al., 1997
; Downward, 1998
). These mutant proteins and the parental oncogenic Ras protein (V12-Ras) were overexpressed in EpH4 cells using respective retroviral constructs (see Materials and methods). Since V12-Ras did not reach similar expression levels in mass cultures as seen for v-H-Ras in EpRas cells (unpublished data), clones were selected expressing particularly high levels of V12-Ras, S35-Ras, and C40-Ras. In Western blots, S35-Ras–expressing clones showed elevated phosphorylation of Erk1 but only basal levels of PKB/Akt phosphorylation compared with empty vector controls. In contrast, the C40-Ras clones showed only basal level phosphorylation of Erks but elevated levels of PKB/Akt phosphorylation ( A). As expected, the V12-Ras control cells showed elevated levels of both phospho-Erk and phospho-PKB/Akt. Similar results were obtained with pools of >10 clones also selected for high level expression of V12-Ras, S35-Ras, and C40-Ras proteins ( B; unpublished data; see Materials and methods).
Figure 4. Biochemical and biological characterization of EpH4 cells expressing Ras effector-specific mutants lacking signaling along the MAPK or PI3K pathways. (A) Clones of EpH4 cells expressing the Ras effector mutants S35-Ras, C40-Ras, or V12-Ras were tested (more ...)
The Ras mutant-overexpressing clones ( A) were then characterized for their response to TGFβ. On porous supports (filters) allowing epithelial polarization, treatment of control V12-Ras cells and two S35-Ras–overexpressing clones with TGFβ for 7 d resulted in spindle-shaped, vimentin-positive, E-cadherin–negative cells ( C; unpublished data). In contrast, C40-Ras–overexpressing cells failed to lose E-cadherin or to upregulate vimentin but showed loss of polarity (redistribution of E-cadherin; C). As expected, control cells not treated with TGFβ always expressed lateral E-cadherin and no vimentin ( C, insets).
In collagen gels, untreated S35-Ras cells resembled V12-Ras cells (e.g., forming distended tubular structures with large lumina in collagen gels), whereas C40 cells more closely resembled EpH4 cells (thin tubules with tiny lumina; A, top). Marker staining revealed basolateral E-cadherin staining and no expression of mesenchymal markers in all three cell types ( B, insets; unpublished data). Treatment of V12-Ras, S35-Ras, and C40-Ras cells with TGFβ resulted in unordered cell strands and cords with spindle-like cellular morphology ( A, middle). After withdrawal of TGFβ, these lumen-less disordered structures persisted in the V12-Ras and S35-Ras cells, whereas C40-Ras cells reverted to thin hollow structures ( A, bottom). Likewise, TGFβ-treated S35-Ras cells showed loss of E-cadherin/β4-integrin staining and induction of the mesenchymal markers vimentin and CD68 ( B, top), a marker distribution persisting after removal of TGFβ ( B, bottom). In contrast, C40-Ras–expressing cells maintained nonpolar E-cadherin staining in the disordered structures induced by TGFβ, whereas mesenchymal markers remained undetectable ( B, top). Upon withdrawal of TGFβ, the C40-Ras structures regained epithelial polarity as indicated by lumen formation and basolateral E-cadherin staining ( B, bottom).
Figure 5. EpH4 cells expressing V12-Ras or S35-Ras undergo EMT upon TGFβ treatment; C40-Ras cells undergo scattering. (A) EpH4 cell clones overexpressing V12-Ras (left) and the Ras effector mutants S35-Ras (middle) and C40-Ras (right; (more ...)
In conclusion, a hyperactive MAPK/Erk pathway plus TGFβ is sufficient to cause EMT, whereas elevated PI3K signaling plus TGFβ induces scattering, that is, spindle-shaped cells without loss of epithelial and gain of mesenchymal markers.
Effector-specific Ras mutants: protection from TGFβ-induced apoptosis requires PI3K signaling
Oncogenic Ras and Raf abolish TGFβ-induced apoptosis in several epithelial cell systems (Oft et al., 1996
; Lehmann et al., 2000
). V12-Ras, S35-Ras, and C40-Ras cells (from both clones and clone pools; A) were therefore tested for their apoptotic response upon TGFβ treatment. This was done using cells pregrown in collagen gels for 3–4 d ( A), since detection of apoptosis was much easier in these cultures than on plastic (Lehmann et al., 2000
). Collagen structures were either collagenase digested and the cells subjected to TUNEL staining in suspension ( C, top) or TUNEL stained and counted in situ, that is, within the intact collagen gels (, bottom; see Materials and methods).
Figure 6. V12-Ras and C40-Ras but not S35-Ras protect EpH4 cells from TGFβ-induced apoptosis. (A) For apoptosis determination in collagen gels, EpH4 cells expressing Ras or Ras effector–specific mutants were allowed to form structures in collagen (more ...)
Both approaches yielded similar results. Already at low TGFβ levels (1 ng/ml), apoptosis of EpH4 control cells was enhanced significantly, whereas >95% of these cells were TUNEL positive and thus apoptotic at 5 ng/ml TGFβ ( C). EpH4 cells expressing V12-Ras and C40-Ras showed no significant elevation of apoptotic cells even at high TGFβ concentrations. In contrast, S35-Ras cells were consistently sensitive to TGFβ-induced apoptosis but required higher TGFβ levels for complete apoptosis than EpH4 cells (). Thus, hyperactivation of the PI3K pathway is required for protection from TGFβ-induced apoptosis. In contrast, hyperactive MAPK signaling in S35-Ras cells failed to induce apoptosis protection at higher TGFβ levels.
Effector-specific Ras mutants in vivo: elevated MAPK and PI3K signaling are each sufficient for tumor formation
To analyze the importance of Ras downstream signaling pathways for tumor formation in vivo, clones strongly overexpressing V12-Ras, S35-Ras, and C40-Ras were injected into the mammary gland fat pads of nude mice. All cell types caused tumors with comparable efficiencies (16/16 injection sites; A). Similar results were obtained with respective pools of clones overexpressing the different Ras signaling mutants except that tumor formation by cells from S35-Ras clone pools was significantly slower than from respective C40-Ras clone pools (unpublished data). Bcl-2–EpH4 control cells formed similarly small regressing nodules as EpH4 cells, which contained only well-polarized, E-cadherin–positive, vimentin-negative cells (unpublished data). Therefore, protection of EpH4 cells from TGFβ-dependent apoptosis is not sufficient for tumorigenesis.
Figure 7. S35-Ras– and C40-Ras–overexpressing cells form tumors, but only S35-Ras cells undergo EMT in vivo. (A) Various clones (see also A) overexpressing V12-Ras (black symbols) S35-Ras (red symbols) and C40-Ras (blue symbols) were injected (more ...)
To address which phenotypic changes of the Ras mutant-expressing cells accompanied tumor growth, we recultivated cells from 4–5-wk-old tumors, using G418 to select for ex-tumor cells of donor origin (see Materials and methods) and analyzed them for E-cadherin and vimentin expression by immunofluorescence. These ex-tumor cells closely resembled the respective Ras mutant-expressing cells subjected to TGFβ treatment in vitro ( C compared with B). V12-Ras and S35-Ras ex-tumor cells showed a typical spindle-shaped morphology (unpublished data) and were vimentin-positive and E-cadherin–negative ( B, left and middle). In contrast, the C40-Ras ex-tumor cells showed epithelioid morphology (unpublished data) and E-cadherin staining, whereas vimentin was undetectable. In conclusion, the separate activation of either the PI3K pathway or the MAPK pathway is sufficient to confer tumorigenic potential to EpH4 cells, whereas EMT in vivo requires the MAPK pathway (see Discussion).
Effector-specific Ras mutants in vivo: only hyperactive MAPK can induce metastasis
Finally, we sought to determine whether or not V12-Ras–, S35-Ras–, or C40-Ras–expressing cells were able to form metastases. In line with metastasis formation in nude mice being a rare event (McClatchey, 1999
), EpRas-derived primary tumors rarely progressed to metastasis before killing the animal. Resection of primary tumors to allow for late metastasis formation met with technical difficulties. Therefore, ex-tumor cells derived from two clones of S35-Ras cells and one clone each of C40-Ras and V12-Ras cells were injected into the tail veins of nude mice, an assay testing for survival of injected cells in the circulation and for evasation and colonization of distant organs. Most of the mice injected with V12-Ras or S35-Ras cells died between 2 and 5 wk, whereas all C40-Ras–injected mice survived until the end of the experiments (10 wk; A). Upon histological analysis, the lungs of dead or moribund animals injected with V12-Ras or S35-Ras cells showed multiple metastases in all cases, often surrounded by blood vessels ( B, left). In contrast, lungs from C40-Ras–EpH4 injected animals were histologically free of metastases ( B, right), and no metastases were found in other organs (unpublished data). Similar results were obtained using pooled clones of V12-Ras–, S35-Ras–, or C40-Ras–EpH4 cells except that the S35-Ras cell-derived metastases appeared much later in line with the slower growth rate of the respective primary tumors (unpublished data).
Figure 8. S35-Ras– but not C40-Ras–overexpressing EpH4 cells form lung metastases upon tail vein injection. (A) Ex-tumor cells from the V12-Ras (black symbols), S35-Ras (red symbols), and C40-Ras clones (blue symbols) indicated were injected intravenously (more ...)
These results indicate that metastasis formation seems to depend entirely on hyperactivity of the Raf/MAPK pathway, whereas a hyperactive PI3K pathway (in C40-Ras cells) is not sufficient for metastasis formation.