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[Cancer Research 62, 4781-4790, August 15, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

MEK1 Signaling Mediates Transformation and Metastasis of EpH4 Mammary Epithelial Cells Independent of an Epithelial to Mesenchymal Transition1

Jan Pinkas and Philip Leder2

Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-mitogen-activated protein kinase (MAPK) pathway is a frequent event in tumorigenesis, and analysis of human breast carcinomas demonstrates that 25–50% of these tumors express elevated levels of activated MAPK1/2. However, a direct role for MEK1 in regulating the invasive and metastatic potential of mammary epithelial cells remains to be established. To directly address the role of constitutive MEK1 signaling in transformation, we have selected the murine mammary epithelial cell line, EpH4, as a model system. EpH4 cells expressing constitutively activated MEK1 display invasive growth in 3-dimensional collagen gels and enhanced motility, and metastatic potential in modified Boyden chamber assays. Furthermore, analysis of markers of normal epithelial morphology by immunofluorescence revealed reorganization of the actin cytoskeleton, and mislocalization of ß-catenin and ZO-1 away from sites of cell-cell contact. However, in contrast to expectations, these changes occurred independently of an epithelial to mesenchymal transition, a change seen frequently in transformed epithelial cells. Moreover, transplantation of EpH4 cells expressing constitutively activated MEK1 into the cleared mammary fat pads of immune-competent hosts rapidly produced tumors that were highly invasive, well vascularized, and readily metastasized to distant organs. Gene expression profiling was performed to identify the downstream targets of MEK1 signaling. Constitutive MEK1 induced the expression of genes involved in proliferation and of matrix metalloproteinases, which regulate invasion and metastasis. These results demonstrate that constitutively activated MEK1 brings about robust tumorigenic changes in murine mammary epithelial cells, and mediates their invasiveness and metastasis in vivo without a requirement for epithelial to mesenchymal transition.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumorigenesis occurs through a stepwise process involving activation of growth-promoting genes and inactivation of tumor-suppressor genes. Many genes that play important roles in these processes have been identified, and analysis of their functions suggests that common pathways involved in proliferation, cell survival, and differentiation are disrupted frequently during tumor development (1 , 2) . Receptor and nonreceptor tyrosine kinases, and the Ras family of GTP-binding proteins are commonly mutated or hyperactivated in the majority of carcinomas, which represent 90% of all human malignancies (3) . One of the most frequent targets downstream of these oncogenes is the MEK3 -MAPK signal transduction pathway (4) . Constitutive activation of the MEK-MAPK pathway has been detected in many human tumors including carcinomas of the breast, colon, kidney, and lung (5, 6, 7, 8) . The growth and invasiveness of ovarian and colon xenograft tumors in vivo was dramatically retarded by pharmacologic inhibition of MEK (9) . In addition, anthrax lethal factor was shown recently to inhibit tumor growth, angiogenesis, and experimental metastasis in an orthotopic tumor model in a MEK-dependent fashion (10) . These experimental results coupled with the frequent activation of MEK in human carcinomas make it an attractive target for cancer therapy, and several MEK-MAPK anticancer drugs are currently in clinical trials (11) . Nonetheless, the molecular alterations associated with MEK-induced transformation of epithelial cells remain poorly defined.

The MEK-MAPK signaling cascade plays an essential role in proliferation and differentiation (12 , 13) . Signal transduction through the MEK-MAPK pathway consists of a series of phosphorylation cascades that connect extracellular stimuli to cytoplasmic and nuclear events. MEK1 is activated downstream of a variety of receptor tyrosine kinases and the Ras family of proto-oncogenes by phosphorylation of two distinct serine residues (14) . Activated MEK1 then phosphorylates specific threonine and tyrosine residues on its only known substrates, MAPK1/2 (15) . Many targets of activated MAPK1/2 have been identified that regulate transcription (16 , 17) , cell survival (18) , and migration (19 , 20) . However, the molecular targets and outcomes of MEK-MAPK signal transduction appear to be very cell type-specific. For example, constitutive activation of MEK1 induced neuronal differentiation of PC12 cells (21) , inhibited muscle differentiation of C2C12 cells (22) , and mediated transformation of fibroblast and epithelial cell lines (21 , 23 , 24) . Recently, a functional proteomics approach using a hematopoietic cell culture model system identified 20 novel targets of MEK-MAPK involved in nuclear transport, nucleosome assembly, and cytoskeletal regulation (25) . This suggests that targets and effectors of MEK signal transduction differ significantly from one another dependent on cell context.

Elevated levels of constitutively activated MEK1 are seen frequently in mouse and human carcinoma cell lines derived from breast, lung, and colon (7 , 9 , 26) . A causative role for constitutive MEK signaling in maintaining the transformed phenotype was suggested through the use of several small molecule inhibitors of MEK activity (9 , 27 , 28) . Studies with these inhibitors demonstrated a role for MEK in mediating enhanced motility (19) , expression of proteinases implicated in invasion and metastasis (29, 30, 31) , and disruption of normal epithelial morphology (32 , 33) . These are common features of the most aggressive and malignant tumor cells, and predict a highly invasive and metastatic phenotype. This morphological transformation is often referred to as EMT, which is characterized by loss of epithelial-specific markers and gain of mesenchymal components (34) . These results suggest that constitutive MEK-MAPK signaling in epithelial cells regulates genes involved in proliferation, invasion, and metastasis, although conclusions drawn from the actions of small molecule inhibitors are difficult to ascribe to a single target.

Several studies examined the transcriptional profile induced by Ras and Raf to identify the molecular targets that might mediate their transforming effects. Analysis of Ha-Ras-transformed rat embryo fibroblasts identified 61 targets that are sensitive to MEK inhibition, which included genes involved in proliferation and modification of the extracellular matrix (31) . More recently, an inducible form of Raf was used to analyze early transcriptional events in a nontransformed human mammary epithelial cell line (35) . Genes involved in proliferation, invasion, and angiogenesis were the most frequent targets identified (35) . Signal transduction downstream of Ras and Raf activates a number of different effectors including other small GTPases and the PI3k pathway, as well as the MEK-MAPK pathway (36) . In addition, evidence exists for Ras/Raf-independent mechanisms of MEK-MAPK activation (37) . Because of these ambiguities, we chose to examine directly the role of constitutive MEK1 signaling in transformation using a murine mammary epithelial cell model.

The EpH4 cell line presents an attractive model in which to study transformation because the cells exhibit morphological and functional characteristics typical of normal mammary epithelial cells. The cells are highly polarized, display a flattened cobblestone morphology, functionally differentiate into complex three-dimensional structures when grown in collagen I gels, and are nontumorigenic on transplantation into athymic nude mice (38, 39, 40) . Previous results demonstrated that EpH4 cells transformed by Ha-Ras (39 , 41) , c-Fos (40) , and c-Jun (38) underwent EMT, but the role of MEK signaling in these processes was not examined. Studies with several murine mammary epithelial cell lines revealed the feasibility of transplanting immortalized cells into the cleared mammary fat pads of syngeneic mice (42, 43, 44, 45) . This suggested that the invasive and metastatic potential of transformed EpH4 cells could be directly assessed in vivo by transplantation into cleared mammary fat pads of immune-competent hosts. In the present study, we demonstrate that constitutive MEK1 signaling morphologically transforms EpH4 mouse mammary epithelial cells. EpH4 cells expressing constitutively activated MEK1 disrupt cell-cell adhesion complexes, reorganize the actin cytoskeleton, and lose the ability to functionally differentiate when resuspended in collagen I gels. Analysis of signal transduction pathways downstream of MEK1 additionally identified components of AP1, cAMP response element, and serum response element transcription factor complexes as being strongly activated. The transcriptional targets of MEK1 were identified by global expression analysis and contain genes involved in proliferation, invasion, and metastasis. Finally, EpH4 cells expressing constitutively activated MEK1 are fully transformed as measured by their ability to rapidly form invasive adenocarcinomas when transplanted into the cleared mammary fat pads of syngeneic mice. Briefly, this study demonstrates that MEK1 transforms normal mammary epithelial cells, and induces a highly invasive and metastatic phenotype in vitro and in vivo without evidence of EMT.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
EpH4 mouse mammary epithelial cells were obtained from Hartmut Beug of the Research Institute of Molecular Pathology (Vienna, Austria). Cells were cultured two-dimensionally on tissue culture plastic (Corning) in DMEM supplemented with 10% bovine calf serum, penicillin-streptomycin (100 µg/ml each), and L-glutamine (2 mM). Cells were cultured three-dimensinoally in collagen I gels as described previously (40) . Briefly, cells from confluent cultures were harvested, counted, and 2 x 104 cells/ml were resuspended in 1 ml of ice-cold collagen I at 1.2 mg/ml (Becton Dickinson) in 12-well tissue culture dishes (Falcon). After the collagen gels had polymerized for 30 min at 37°C, they were overlayed with 1 ml of MEGM (Clonetics) that consists of basal medium supplemented with bovine pituitary extract (52 µg/ml), hydrocortisone (0.5 µg/ml), epidermal growth factor (10 ng/ml), insulin (5 µg/ml), gentamicin (50 µg/ml), and amphotericin B (50 ng/ml).

Vectors and Transfections.
To target high level expression in EpH4 cells a new expression construct was created. The CMV IE enhancer/chicken ß-actin promoter was excised from pCAGGS (46) with XhoI and EcoRI, and cloned into a derivative of pcDNA3 (Invitrogen). The pcDNA3 vector was modified by excising the CMV promoter and multiple cloning site with BglII and XhoI, and ligating in an oligonucleotide linker containing XhoI, KpnI, HindIII, EcoRI, BamHI, BglII, NotI, and ClaI restriction sites to create pcDNA3-LINKER. The CMV IE enhancer/chicken ß-actin promoter was then ligated into the XhoI and EcoRI sites of pcDNA3-LINKER to create pß. The new vector (pß) uses the strong chicken ß-actin promoter to drive transgene expression, contains an extended multiple cloning site, and has a neomycin resistance cassette for selection in eukaryotic cells. The Glu-Glu epitope-tagged phosphorylation site MEK1 mutant (MEKDD) was excised from pGEM7-MEKDD (47) and cloned into pß. The pß and pß-MEKDD constructs were linearized with PvuI and then transfected into EpH4 cells using Fugene-6 (Roche). After selection in complete medium supplemented with 1 mg/ml G418 (Invitrogen/Life Technologies, Inc.) for 14 days, individual clones were isolated and expanded.

Western Blotting.
EpH4 cells grown in complete medium were washed with PBS, and then lysed in 150 mM NaCl, 50 mM Tris (pH 7.6), 10 mM sodium PPi, 10 mM NaF, 2 mM EDTA, 1% NP40, 0.1% SDS, 0.5% deoxycholate, 1 mM sodium orthovanadate, 10 mM ß-glycerophosphate, and Complete protease inhibitor tablets (Roche). Protein concentration in the lysates was measured using Bradford reagent (Bio-Rad) with BSA as a standard. Equivalent amounts of protein were separated by SDS-PAGE on 10% polyacrylamide gels and then transferred to Immobilon-P membranes (Millipore Corporation) using a Hoeffer SemiPhor transfer apparatus. Western blotting was performed according to the manufacturer’s protocols with the following antibodies: monoclonal anti-ß-actin (AC-15), monoclonal anti-{alpha} smooth muscle actin (1A4), and monoclonal anti-vimentin (VIM 13.2) from Sigma; monoclonal anti-E-cadherin from Transduction Laboratories; polyclonal anti-CREB, polyclonal anti-phospho-CREB(Ser133), polyclonal anti-c-Jun, polyclonal anti-phospho-c-Jun(Ser63)II, and polyclonal anti-phospho-p44/42 MAPK(Thr202/Tyr204) from Cell Signaling Technology; polyclonal anti-extracellular signal-regulated kinase 1(C-16) from Santa Cruz; monoclonal anti-cytokeratin 18 (Ks 18.04) from Research Diagnostics Inc.; and monoclonal anti-Glu-Glu from Covance. Proteins were visualized using a horseradish peroxidase-linked secondary antibody (Jackson ImmmunoResearch) and a chemiluminescent detection system (enhanced chemiluminescence; Amersham).

Boyden chamber Assays.
Migration and invasion assays were performed using modified Boyden chambers with filter inserts for 24-well dishes containing 8-µm pores (Becton Dickinson) as described previously (48) . Uncoated filters were used for migration assays, and Matrigel coated filters were used for invasion assays. Cells (1 x 105) were plated into 300 µl of MEGM in the upper chamber, and the lower chamber was filled with 500 µl of MEGM. After 24 h in culture, cells were fixed in 2.5% glutaraldehyde in PBS for 5 min and then stained with 0.5% toluidine blue in 2% Na2CO3 for 5 min. Cells on the upper side of the filters were removed with cotton-tipped swabs, and the filters were washed in PBS. Cells on the underside of the filters were counted by microscopic inspection. Each clone was plated in triplicate per experiment, and each experiment was repeated at least three times.

Immunofluorescence.
EpH4 cells grown on four-chambered plastic slides (Nunc) were fixed in 3.7% formaldehyde in PBS for 5 min, permeabilized with 0.5% Triton X-100-PBS for 5 min, and then stained with 2 units/ml Alexa594-phalloidin (Molecular Probes) for 30 min to visualize polymerized actin. EpH4 cells were costained with mouse monoclonal anti-Glu-Glu antibody (Covance; 1:500 dilution), mouse monoclonal anti-ß-catenin antibody (Transduction Laboratories; 1:100 dilution), or rabbit polyclonal anti-ZO-1 antibody (Zymed; 1:100 dilution) in PBS for 1 h at room temperature. Secondary staining was performed with FITC-conjugated donkey antimouse or antirabbit antibody (Jackson ImmunoResearch; 1:100 dilution) in PBS for 1 h at room temperature. The slides were washed in PBS, dipped in H2O, and mounted under glass coverslips in 15% Gelvatol (polyvinyl alcohol), 33% glycerol, and 2.5% diazobicyclo-octane (Sigma) in PBS.

Luciferase Reporter Assays.
Transient transfections were performed in six-well tissue culture dishes (Corning) using Fugene-6 according to the manufacturer’s protocol. The following luciferase reporter constructs were used: pp53-LUC, pCRE-LUC, pSRE-LUC, and pAP1-LUC (all from Stratagene). A ß-galactosidase control vector, pCMV-ßGAL (49) , was used to control for differences in transfection efficiency. Transfections were performed in triplicate with 1 µg luciferase reporter/0.25 µg pCMV-ßGAL, and cells were harvested 24 h after transfection. Lysates were prepared using 150 µl of Passive Lysis Buffer (Promega) according to the manufacturers protocol, and samples were analyzed on an Automat LB953 luminometer (Berthold) with automatic injection of luciferase reagent (Promega). ß-Galactosidase activity was determined using the Galacto-Star kit (Tropix) with signal detection performed using a 1209 RackBeta liquid scintillation counter (LKB Wallac). Luciferase activities were normalized to ß-galactosidase activity to control for differences in transfection efficiency.

Global Expression Analysis.
Total RNA from EpH4 ß2 and ßMEKDD-47 cells was isolated, and cDNA probes were synthesized using the Atlas Pure Total RNA Labeling System (Clontech). Atlas Mouse 1.2 arrays were hybridized according to the manufacturer’s protocol (Clontech) and then were exposed to BioMax MS autoradiography film (Kodak) at -70°C for 8–72 h. Autoradiographs of similar exposure were scanned on a flatbed scanner and analyzed using Atlas Image 2.0 software (Clontech). Each sample was used to probe duplicate arrays, and only signals that were reproducibly expressed on both arrays were examined further. The data in Table 1Citation are a pair-wise comparison between one representative autoradiograph each of EpH4 ß2 and ßMEKDD-47 hybridized arrays. Because of the limited dynamic range of the autoradiographic film, several signals are denoted as either "UP," detectable signal in film #1 and background signal in film #2, or "DOWN," detectable signal in film #2 and background signal in film #1. Only signals that changed >2.5-fold or that were "UP" or "DOWN" were considered to be significantly different between the two samples and were examined further.


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  		Table 1 Differential gene expression in EpH4 ß2 vs. ßMEKDD-47 cells

 
RNA Extraction and Northern Blot Analysis.
Total RNA was isolated from subconfluent EpH4 cells using Ultraspec-II and RNATack Resin according to the manufacturer’s protocol (Biotecx). Total RNA (10 µg) was separated by denaturing agarose/formaldehyde gel electrophoresis and transferred to GeneScreen Plus membranes (NEN Life Science Products). Fragments of the murine cDNAs for c-Src, gelatinase-B, ID3, Wnt-7A, placental and embryonic homeobox (PEM), and actinwere amplified by reverse transcription-PCR from EpH4 total RNA. The identity of the amplified cDNAs was confirmed by double-stranded DNA sequencing. cDNA fragments were labeled with [{alpha}-P32]dCTP using the Prime-It II labeling kit (Stratagene), and Northern blots were hybridized at 42°C for 16 h. Signals were detected by autoradiography followed by PhosphorImager analysis using a Storm860 phosphorimager (Molecular Dynamics).

Mammary Gland Transplantation.
BALB/c mice were obtained from Charles River Labs and were maintained according to Institutional Animal Care and Use Committee-approved guidelines. EpH4 cells were transplanted into the cleared number 4 mammary fat pads of 21–25 day-old female syngeneic BALB/c mice by standard procedures (50) . Briefly, EpH4 cells were grown to confluence, harvested, resuspended in PBS, and then 5 x 105 cells/10 µl were injected into both cleared number 4 mammary fat pads distally to the lymph node using a Hamilton syringe. Starting at 1 week after transplantation, the mice were palpated biweekly for mammary tumors. EpH4 ßMEKDD transplants gave rise to palpable mammary tumors within 7–10 days, and the animals were sacrificed 5–7 weeks after transplant with mammary tumors 1–2 cm3. EpH4 control and ß2 transplanted mammary fat pads were either isolated at 8 weeks after transplant to assess outgrowth or were observed for >6 months to monitor long-term tumor development. Tissues were fixed in 10% neutral-buffered formalin (Fisher), and were processed for routine H&E staining and histopathology. Cell lines were rederived from EpH4 ß2 transplants, and ßMEKDD primary tumors and metastases by sterilely dissecting a portion of the outgrowth, disaggregating the cells, and selecting in complete medium supplemented with 1 mg/ml G418.

Inverted and Fluorescence Microscopy and Photography.
All of the cells grown on tissue culture plastic and in collagen I gels were observed using a Zeiss Axiovert25 inverted microscope, and images were captured with a Nikon D1 digital camera. All of the fluorescence experiments were observed using a Zeiss Axioskop upright fluorescence microscope with the appropriate fluorescence filter sets, and images were captured using a SPOT-RT cooled digital camera (Diagnostic Instruments).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Constitutively Activated MEK1 Induces Morphological Features of Transformation in EpH4 Cells.
To address the role of the MEK-MAPK pathway in transformation, constitutively activated MEK1 was stably expressed in EpH4 (40) murine mammary epithelial cells. Activation of MEK1 is mediated through phosphorylation of serines at positions 218 (Ser218) and 222 (Ser222) by members of the Raf (MEK kinase) family of kinases (14) , and substitution of Ser218/Ser222 with either aspartic or glutamic acid mimics the phosphorylated and activated state (47) . The Asp218/Asp222 MEK1 phosphorylation site mutant (MEKDD) was shown to be the most potent in terms of its ability to activate MAPK1/2 and to transform NIH-3T3 cells (47 , 51) . We generated EpH4 clones stably expressing an epitope-tagged constitutively activated form of MEK1 (MEKDD-Glu-Glu). Western blot analysis identified multiple clones that expressed high levels of Glu-Glu-tagged MEKDD (Fig. 1A)Citation . Indirect immunofluorescence using an antibody recognizing the Glu-Glu epitope tag demonstrated that all of the cells in the ßMEKDD clones expressed constitutively activated MEK1 (Fig. 2, B and CCitation ; data not shown), which was primarily localized in the cytoplasm as expected (52) . The activity of MEKDD in these clones was measured by determining the levels of phosphorylated MAPK1/2 in exponentially growing cells. Elevated levels of phosphorylated MAPK1/2 are seen in EpH4 ßMEKDD clones as compared with parental or vector control cells (Fig. 1B)Citation in agreement with previous results from fibroblast cell lines (47) .



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  		Fig. 1. Expression of constitutively activated MEK1 alters cell morphology and blocks functional differentiation in collagen I gels. Western blot analysis of (A) Glu-Glu-tagged MEKDD, (B) phospho-MAPK1/2, (C) MAPK1/2, and (D) ß-actin protein levels in EpH4 parental, vector control, and four independent clonal ßMEKDD cell lines. E, phase-contrast photomicrographs of parental EpH4 and two independent clonal ßMEKDD cell lines growing at low density on tissue culture plastic. bar, 10 µM. F, phase-contrast photomicrographs of parental EpH4 and two independent clonal ßMEKDD cell lines growing resuspended in collagen I gels after 12 days in culture. bar, 20 µM.

 


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  		Fig. 2. Constitutively activated MEK1 disrupts cell-cell junctions and reorganizes the actin cytoskeleton in EpH4 mammary epithelial cells. The cells were fixed with formaldehyde and stained for filamentous actin with (A'–I') Alexa594-Phalloidin, and then immunostained for (A–C) Glu-Glu, (D–F) ß-catenin, and (G–I) ZO-1. Each vertical pair of panels (i.e., A and A') represents an identical field of cells. bar, 10 µM.

 
Cells expressing constitutively activated MEKDD exhibited distinct morphological changes when grown on tissue culture plastic as compared with parental cells (Fig. 1E)Citation , but did not demonstrate any difference in growth rate in complete medium and were not able to grow serum independently (data not shown). EpH4 cells display a cobblestone morphology typical of nontransformed polarized epithelial cells, and MEKDD-expressing cells appeared to have disrupted cell-cell junctions, were more refractile, and appeared to be highly motile (Fig. 1E)Citation . These morphological defects are characteristic of transformed cells and could be reverted with the MEK-specific inhibitor U0126 (28) in a concentration-dependent manner (data not shown). Parental EpH4 cells form branching ductal structures when grown in three-dimensional collagen I gels in defined medium (Fig. 1F)Citation in agreement with previous results (39 , 53) . However, the MEKDD clones lost the ability to functionally differentiate and grew as invasive single cells when resuspended in collagen I gels (Fig. 1F)Citation .

Disruption of cell-cell adhesion and integrity of intercellular junctions correlates strongly with increased invasiveness and motility of tumor cells (34 , 54) . Therefore, we determined whether constitutively activated MEK1 affects morphological markers of cell-cell adhesion in EpH4 cells. Typical of polarized epithelial cells, EpH4 parental and ß2 cells localize ß-catenin and ZO-1 to sites of cell-cell contact (Fig. 2, D and G)Citation , and bundle actin to the cell periphery (Fig. 2A', 2D', and 2G')Citation . These results are consistent with previous reports examining markers of normal epithelial morphology in EpH4 cells (38, 39, 40 , 55) . However, MEKDD-expressing cells exhibited reorganized actin microfilaments (Fig. 2B', 2C', 2E', 2F', 2H', and 2I')Citation , and mislocalized ß-catenin and ZO-1 away from cell-cell junctions (Fig. 2E, 2F, 2H, and 2ICitation ; data not shown). The phenotype of the EpH4 ßMEKDD clones growing on tissue culture plastic and in collagen gels suggests that the cells are more migratory and/or invasive than parental EpH4 cells. To quantify the migratory and invasive capacity of the cells, modified Boyden chamber assays were performed. EpH4 ßMEKDD cells exhibited a 4–6-fold increased rate of migration through uncoated Boyden chambers as compared with the parental cells or ß2 cells (Fig. 3A)Citation . Movement of cells through Matrigel-coated Boyden chambers mimics the early steps of tumor invasion in vivo by requiring proteolytic degradation of the basement membrane before cells can gain access to the pores in the filter inserts. MEKDD-expressing cells exhibited an 8–10-fold increased rate of invasion as compared with parental EpH4 or ß2 cells (Fig. 3B)Citation . These results demonstrate that EpH4 ßMEKDD cells exhibit in vitro markers of transformation and suggest that they will be tumorigenic and invasive in vivo.



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  		Fig. 3. EpH4 cells expressing constitutively activated MEK1 are more migratory and invasive in modified Boyden chamber assays in vitro. EpH4 parental, vector control, and four independent ßMEKDD lines that had (A) migrated through uncoated filter inserts or (B) invaded through Matrigel coated filter inserts were fixed, stained, and counted. Each experiment was performed in triplicate, and the values represent the average; bars, ±SD.

 
Downstream Targets of Constitutively Activated MEK1 in EpH4 Mammary Epithelial Cells.
Signal transduction pathways and molecular targets modulated downstream of MEK1 are highly cell type-specific (12) . It has been shown previously in a number of different cell types that MEK1 signaling results in activation of components of AP1, CRE, and SRE transcription factor complexes (16 , 17) . To identify signaling pathways downstream of constitutively activated MEK1 in EpH4 mammary epithelial cells, transcription-based luciferase reporter assays were performed. EpH4 ßMEKDD clones demonstrate strong transcriptional activation through AP1, CRE, and SRE DNA-binding motifs but not through the p53 response element (Fig. 4A)Citation or a minimal promoter (data not shown). This demonstrates that constitutively activated MEK1 regulates specific transcriptional events in EpH4 cells.



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  		Fig. 4. Components of AP1, CRE, and SRE transcription factor complexes are activated in EpH4 ßMEKDD cells. A, transcriptional activation downstream of MEK1 was determined by transient transfection of AP1, CRE, SRE, and p53 luciferase reporter constructs into EpH4 control and ßMEKDD cells. The luciferase values were normalized to a constitutively expressed cotransfected ß-galactosidase vector to control for differences in transfection efficiency. Each experiment was performed in triplicate, and the values represent the average; bars, ±SD. Western blot analysis of (B) phospho-c-Jun, (C) c-Jun, (D) phospho-CREB, (E) CREB, and (F) ß-actin protein levels in EpH4 parental, vector control, and four independent clonal ßMEKDD cell lines.

 
To identify the components of these transcription factor complexes, Western blot analysis was performed using antibodies that can distinguish between total and phosphorylated forms of these proteins. Phosphorylated MAPK1/2 translocate into the nucleus where they can phosphorylate and, thus, activate components of these transcription factor complexes (12) . EpH4 ßMEKDD cells express elevated levels of total c-Jun protein (Fig. 4C)Citation as well as phosphorylated and activated c-Jun as compared with control cells (Fig. 4B)Citation . The expression of phosphorylated CREB (Fig. 4D)Citation is also induced in the MEKDD-expressing cells without any significant change in total CREB protein levels (Fig. 4E)Citation . However, there is no change in the activation of other MAPK signaling cascades such as the c-Jun NH2-terminal kinase and p38 pathways or in the activity of Akt, which is downstream of PI3k (data not shown). These results corroborate the luciferase reporter data and demonstrate that signal transduction downstream of MEK1 activates specific components of AP1 and CRE transcription factor complexes without affecting other signal transduction pathways.

Global expression analysis was performed to identify genes that are induced or repressed in response to constitutive MEK1 signaling in EpH4 mammary epithelial cells. Clontech Atlas Mouse 1.2 arrays contain 1176 cDNAs representing a broad spectrum of genes involved in normal developmental processes and transformation.4 The arrays were hybridized with labeled cDNA from EpH4 ß2 and ßMEKDD-47 cells in duplicate and then analyzed using AtlasImage 2.0 software. Pair-wise analysis of duplicate arrays hybridized with labeled cDNA from the same cell source demonstrate that the expression profiles are highly reproducible across independently probed arrays (data not shown). Analysis of arrays hybridized with labeled cDNA from EpH4 ß2 versus ßMEKDD-47 cells identify only 19 genes of which the expression changes >2.5-fold out of >300 genes expressed (Table 1)Citation . Included in this list are genes involved in proliferation, invasion, and metastasis. This demonstrates that constitutive MEK1 signaling modulates the expression of a limited number of downstream targets.

To validate the results from the arrays, Northern blot analysis was performed on RNA isolated from EpH4 parental, ß2, and four independent clonal ßMEKDD cell lines. Probes for a subset of genes identified as being significantly different on the arrays were amplified by reverse transcription-PCR and then used to probe Northern blots. The results from the Atlas Mouse 1.2 arrays correlate well with data from Northern blot analyses demonstrating that 9 of 12 genes exhibit similar expression patterns and fold-change (Fig. 5A–ECitation ; Table 1Citation ; data not shown). The expression patterns in the independent ßMEKDD clones are consistent suggesting that these genes represent primary and conserved secondary targets of constitutively activated MEK1. Interestingly, several proteases (Gelatinase-B, UPA, and UPAR1) that have been shown previously to play a role in increased invasiveness, and metastasis of human breast carcinoma cell lines are strongly induced (19 , 30) . This provides a mechanistic explanation for the increased in vitro metastatic potential of the cells (Fig. 3B)Citation and suggests that the EpH4 ßMEKDD clones will be invasive and metastatic in vivo.



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  		Fig. 5. Expression analysis of MEKDD target genes in EpH4 cells. RNA from EpH4 parental, vector control, and four independent clonal ßMEKDD cell lines was separated by denaturing electrophoresis, blotted onto charged membranes, and probed with labeled cDNAs for (A) c-Src, (B) ID3, (C) PEM, (D) Gelatinase-B, (E) Wnt-7A, and (F) actin.

 
Transplantation of EpH4 Cells into Cleared Mammary Fat Pads.
Murine mammary epithelium has the capacity to recapitulate normal ductal development on transplantation into the cleared mammary fat pad of a syngeneic host (50) . More recently, immortalized mammary epithelial cell lines were shown to produce outgrowths when transplanted into cleared mammary fat pads (42, 43, 44, 45) . This capacity for outgrowth was exploited to examine the role of MEK1 in tumorigenesis using EpH4 mammary epithelial cells. EpH4 parental, ß2, and ßMEKDD cells were transplanted (5 x 105 cells/10 µl) into cleared number 4 mammary fat pads of syngeneic BALB/c hosts and were monitored for the development of tumors. Mammary fat pads transplanted with EpH4 parental and ß2 cells never produced mammary tumors over a 4–6 month observation period (Table 2)Citation . On the other hand, EpH4 ßMEKDD cells rapidly formed palpable lesions within 7–10 days after transplantation that went on to produce highly invasive tumors of 1–2 cm3 by 5–7 weeks (Table 2)Citation . H&E-stained sections of these outgrowths reveal that the EpH4 ßMEKDD cells form poorly differentiated adenocarcinomas that had completely filled the fat pad. These lesions are well vascularized and have areas of dense fibrosis (Fig. 6C)Citation . The kinetics of tumor development was highly reproducible with 73 of 74 transplants representing four independent ßMEKDD cell lines producing outgrowths that rapidly progressed to invasive adenocarcinomas. In contrast, the parental EpH4 and ß2 control cells formed limited outgrowths in the vicinity of the transplant site and did not fill the fat pad. The control outgrowths produced small cystic structures reminiscent of alveolar development (arrowheads in Fig. 6BCitation ) but did not recapitulate normal ductal development. The lobular morphology of the outgrowths was not unexpected, because EpH4 cells were derived from mid-pregnant mammary epithelium (40 , 56) . These data demonstrate that EpH4 cells expressing constitutively activated MEK1 are fully transformed and tumorigenic in vivo.


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  		Table 2 Tumor development in EpH4 mammary fat pad transplants

 


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  		Fig. 6. Histopathology of EpH4 control and ßMEKDD mammary fat pad transplants. H&E-stained sections of (A) cleared mammary fat pad, (B) EpH4 ß2, and (C) EpH4 ßMEKDD-47 mammary outgrowths. Arrowheads in B mark the cystic outgrowths seen in EpH4 control and ß2 mammary transplants. Note the highly vascular nature of the EpH4 ßMEKDD-47 mammary tumor (C). bar, 10 µM.

 
To determine whether growth of EpH4 cells in vivo selected for clonal variants that were significantly different from cells before transplant, we excised mammary tumors from ßMEKDD transplants and ß2 outgrowths, and rederived cell lines by selection in G418. Drug-resistant cell lines were readily re-established from seven of seven mammary tumors representing four ßMEKDD clones and from two of two ß2 outgrowths. Western blot analysis demonstrated that the rederived tumor cell lines maintained high levels of Glu-Glu-tagged MEKDD (Fig. 7A)Citation and activated MAPK1/2 (Fig. 7B)Citation that were comparable with the ßMEKDD clones before transplant (Fig. 1A)Citation . These results indicated that the rederived cells originated from the transplanted cells and that transgene expression was required for tumor development. The invasive nature of the mammary tumors coupled with data from in vitro Boyden chamber assays (Fig. 3B)Citation and global expression analysis (Table 1)Citation suggested that the EpH4 ßMEKDD cells were metastatic. To identify metastatic cells in animals carrying ßMEKDD mammary tumors, various internal organs were isolated at sacrifice, disaggregated, and placed under G418 selection. Metastatic cell lines were rapidly reisolated from lung, spleen, and kidney in five of seven animals carrying primary mammary tumors but not from animals transplanted with EpH4 ß2 cells (Table 2)Citation . Indirect immunofluorescence using an antibody that recognizes the Glu-Glu epitope tag demonstrated that all of the cells rederived from primary tumors and metastases expressed constitutively activated MEKDD (Fig. 7, F and GCitation ; data not shown). These data demonstrated that EpH4 ßMEKDD cells are metastatic in vivo, and the induction of matrix metalloproteinases provides a mechanistic explanation for the increased metastasis.



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  		Fig. 7. Rederived tumor cells lines maintain MEKDD expression and activation of downstream targets. Western blot analysis of (A) Glu-Glu-tagged MEKDD, (B) phospho-MAPK1/2, (C) MAPK1/2, and (D) ß-actin protein levels in EpH4 parental and ßMEKDD tumor cell lines. The cells were fixed with formaldehyde and stained for (E'–G') filamentous actin with Alexa594-phalloidin, and then immunostained for (E–G) Glu-Glu-tagged MEKDD. Each vertical pair of panels (i.e., E and E') represents an identical field of cells. bar, 10 µM.

 
It has been shown previously that transformation of EpH4 cells by Ha-Ras (39 , 41) and MDCK cells by Raf (57) strongly induce TGF-ß expression to mediate EMT and increase malignancy. Our morphological data suggested that the EpH4 ßMEKDD cells and reisolated tumor cells had undergone EMT. Indirect immunofluorescence revealed severe disorganization of the actin cytoskeleton (Fig. 7, F' and G')Citation , and mislocalization of ß-catenin and ZO-1 away from sites of cell-cell contact (data not shown) as compared with a cell line reisolated from an EpH4 ß2 outgrowth (Fig. 7, E and E'Citation ; data not shown). Markers specific for epithelial and mesenchymal cells were examined to determine whether the ßMEKDD cells had undergone an EMT. Western blot analysis indicated that the cells maintained expression of epithelial specific genes, E-cadherin and keratin 18 (Fig. 8, A and B)Citation and did not induce mesenchymal-specific genes such as smooth muscle actin and vimentin (Fig. 8, C and D)Citation . In addition, expression of constitutively activated MEK1 did not induce expression of TGF-ß or alter the response to growth inhibitory concentrations of TGF-ß (data not shown). These data indicate that constitutively activated MEK1 is sufficient to transform EpH4 mammary epithelial cells independent of TGF-ß-mediated EMT.



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  		Fig. 8. EpH4 ßMEKDD and tumor-rederived cell lines have not undergone EMT. Western blot analysis of (A) E-cadherin, (B) keratin 18, (C) smooth muscle actin, (D) vimentin, and (E) ß-actin protein levels in EpH4 control and ßMEKDD cell lines (left) and in rederived tumor cell lines (right). Protein lysates from NIH-3T3 fibroblasts were included in each panel as a positive control for mesenchymal specific markers.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The MEK-MAPK signal transduction pathway is activated frequently during human breast tumorigenesis (5, 6, 7) , and expression correlates with lymph node involvement and metastasis (8) . Recent studies with small molecule inhibitors have demonstrated the importance of MEK1 signaling in mediating the transformed phenotype in orthotopic tumor transplant models (9 , 10) , and MEK inhibitors are currently in clinical trials as potential cancer therapeutics (11) . However, a direct role for MEK1 in regulating the invasive and metastatic potential of mammary epithelial cells remains to be established. We have used EpH4 cells (40) , an immortalized murine mammary epithelial cell line, to directly address this issue. Stable expression of constitutively activated MEK1 in EpH4 cells results in the disruption of morphological markers typical of polarized epithelial cells but does not affect the rate of proliferation in medium with or without serum (data not shown). This is consistent with previous results from fibroblast and epithelial cell lines demonstrating that constitutive MEK1 signaling is sufficient to induce morphological changes indicative of transformation but is not sufficient to promote serum-independent growth (58, 59, 60) . The disruption of normal epithelial morphology has been shown to correlate strongly with increased malignancy and invasiveness of tumor cells (3 , 61) . To examine the metastatic potential and rate of migration in vitro we used modified Boyden chamber assays. The data demonstrated that EpH4 ßMEKDD cells exhibited increased motility and metastatic potential in vitro as compared with control cells. Whereas previous studies have found a correlation between activation of the MEK-MAPK pathway and in vitro motility and invasiveness, our results demonstrate directly that MEK1 is sufficient to induce these metastatic characteristics (29, 30, 31) . The increased metastatic potential of EpH4 ßMEKDD cells in vitro in Boyden chamber assays and in collagen I gels suggest that a strong induction of matrix metalloproteinases mediates degradation of the extracellular matrix.

Analysis of markers of normal epithelial morphology by immunofluorescence revealed mislocalization of ß-catenin and ZO-1 away from sites of cell-cell adhesion, and reorganization of the actin cytoskeleton in EpH4 cells expressing constitutively activated MEK1. Previous studies have shown that morphological transformation of epithelial cells is often accompanied by an EMT (34) . Specifically, studies with mammary epithelial cells demonstrated that transformation of EpH4 cells by Ha-Ras (39 , 41) , c-Jun (38) , and c-Fos (40) and MCF-10 cells by Raf (35 , 57) resulted in severe disruption of cell morphology and induction of EMT. However, the role of MEK1 signaling in mediating the changes in cell morphology and the possible uncoupling of EMT and malignant transformation had not been demonstrated. In the present study, we addressed this question directly, and our in vitro data demonstrate that disruption of cell morphology by constitutively activated MEK1 occurred independently of an EMT. EpH4 cells expressing constitutively activated MEK1 did not repress expression of epithelial-specific genes like E-cadherin and keratin 18 or induce expression of mesenchymal-specific genes such as vimentin and smooth muscle actin. These data clearly demonstrated that constitutive MEK1 signaling is not sufficient to induce EMT of mammary epithelial cells. However, it has yet to be determined whether MEK1 signaling is required for EMT induced by Ras or Raf in mammary epithelial cells. Recently, studies with MDCK cells transformed by Ras demonstrated that pharmacologic inhibition of MEK1 restored normal epithelial morphology and E-cadherin expression levels (32) . These data suggest but do not directly demonstrate that constitutive MEK1 signaling is required for Ras-mediated EMT. We hypothesize that additional pathways downstream of Ras and Raf, such as PI3k, in addition to MEK-MAPK are required for this additional step.

A role for EMT in human breast tumorigenesis remains difficult to establish conclusively. Primary breast tumors are heterogeneous at the cellular level with components derived from luminal epithelial, myoepithelial, and mesenchymal cell types (62) . Conclusive evidence for EMT will require the identification of additional markers to distinguish between myoepithelial cells, myofibroblasts, and epithelial to mesenchymal-derived cells. In addition, a careful analysis of constitutive MEK/MAPK signaling and multiple markers of EMT has not yet been performed in the same panel of primary and metastatic breast cancers.

Global expression analysis was performed to identify genes differentially expressed in response to constitutively activated MEK1 in EpH4 cells. Recently, the transcriptional profile of a hormone-activatable isoform of Raf was examined in a human mammary epithelial cell line (35) . Few of the transcriptional changes identified by Schulze et al. (35) were reproduced in our studies. This discordance most probably resulted from several fundamental differences in experimental design between the two studies. The study of Schulze et al. (35) concentrated on immediate early transcriptional changes induced by Raf in growth factor-depleted medium. On the other hand, we screened for genes differentially expressed in response to constitutive MEK1 signaling in cells growing in complete medium supplemented with serum. Genes identified in our study most probably represent primary as well as secondary targets involved in the establishment and maintenance of the transformed state. One other study examined MEK-dependent transcriptional targets in a Ras transformed fibroblast cell line (31) . Very few of the differentially expressed genes identified by Zuber et al. (31) were expressed in a similar fashion in the current study. We believe that is because of the different cell types used for each study, i.e., fibroblast versus epithelial. In addition, signal transduction downstream of Ras and Raf will activate other effector pathways as well as MEK-MAPK.

Many transcriptional regulators were differentially expressed in the EpH4 ßMEKDD cells. However, a role for several of these factors (EN1, HNF4, HOXD4, ID3, Kreisler, and PEM) in the induction or maintenance of the transformed state remains unclear. Previous studies with Raf-transformed MDCK cells (57) and Ha-Ras-transformed EpH4 cells (39 , 41) demonstrated a marked induction of TGF-ß expression at the mRNA and protein levels. We have examined the expression levels of TGF-ß, TGF-ßRI, and TGF-ßRII in EpH4 ßMEKDD cells, and were unable to demonstrate any significant differences from the control cells (data not shown). This suggested that EpH4 ßMEKDD cells remain sensitive to the effects of TGF-ß, and preliminary evidence demonstrates that EpH4 ßMEKDD cells retain their sensitivity to TGF-ß-mediated growth inhibition and do not undergo EMT after prolonged treatment with TGF-ß (data not shown). However, several TGF-ß-related genes were differentially expressed (BMP7, Inhibin ßE, and Endoglin) in EpH4 ßMEKDD cells. This suggests that a noncanonical TGF-ß pathway may be activated in response to constitutive MEK1 signaling in mammary epithelial cells, and we are currently examining this possibility.

Interestingly, several proteases (UPA, UPAR1, and Gelatinase-B) involved in invasion and metastasis were up-regulated in ßMEKDD cells. The induction of matrix metalloproteinases provides a mechanistic explanation for the increased metastatic potential seen in Boyden chamber assays in vitro and invasive growth in collagen I gels. This correlated well with data from human breast carcinoma cell lines demonstrating that constitutive MEK1 signaling mediated increased invasiveness and metastatic potential in vitro (19 , 30) . Expression of constitutively activated MEK1 was shown recently to associate strongly with decreased disease-free survival and increased lymph node involvement in a panel of human breast carcinomas (8) . Taken together, these data allow us to predict that EpH4 ßMEKDD cells will be highly invasive and metastatic in vivo.

Previous results with COMMA-D and HC11 mouse mammary epithelial cells lines demonstrated the feasibility of fat pad transplantation as a model for mammary tumorigenesis (43, 44, 45) . This approach appears to be a significant improvement over mammary fat pat transplantation of retrovirally infected primary mammary epithelial cells. Infection of cells with an ERBB2/NEU-expressing retrovirus resulted in only a sporadic tumor phenotype with a long latency (45 , 63 , 64) . The low rate of infection and moderate expression levels of the transgene most probably are the cause of the weak tumor phenotype. However, EpH4 mammary fat pad transplantation has limitations as well. In contrast to transplanted primary mammary epithelial cells, parental EpH4 cells did not recapitulate a normal mammary developmental architecture when reintroduced into cleared mammary fat pads. This data are consistent with previous studies demonstrating that outgrowths from mammary fat pad transplants of a series of immortalized mammary epithelial cell lines formed aberrant ductal and/or alveolar structures (42, 43, 44) . Nevertheless, the rapid and reproducible kinetics of tumor development make EpH4 mammary fat pad transplants an attractive in vivo model for transformation, metastasis, and screening of cancer therapeutics in immune-competent hosts.

To confirm the transformed phenotype seen in vitro, we transplanted cells expressing constitutively activated MEK1 into cleared mammary fat pads of syngeneic BALB/c mice. EpH4 ßMEKDD cell transplants rapidly generated invasive tumors, but control cells never produced any malignant outgrowths. A direct role for constitutive MEK1 signaling in mediating the transformed phenotype was confirmed by demonstrating that the primary tumors and rederived cell lines expressed high levels of Glu-Glu-tagged MEKDD. Histological analysis of the tumors revealed that they were poorly differentiated adenocarcinomas characterized by stromal reactivity and high mitotic index. The outgrowths quickly filled the entire fat pad, and frequently invaded adjoining muscle and the abdominal cavity (data not shown). The rapid growth of the tumors was most probably supported by the intense neovascularization evident throughout the outgrowths. It was demonstrated recently in a transgenic pancreatic tumor model that expression of Gelatinase-B regulated an angiogenic switch during tumor development (65) . The induction of Gelatinase-Bin the EpH4 ßMEKDD cells might play a similar role in promoting angiogenesis in the outgrowths in vivo. Finally, we demonstrated that mammary tumors from EpH4 ßMEKDD outgrowths readily metastasized to distant organs in immune-competent hosts. The metastatic phenotype extends previous results demonstrating that constitutively activated MEK1 enhanced lung colonization of NIH-3T3 cells in an experimental metastasis model system in athymic nude mice (51) . In conclusion, we demonstrate that constitutive MEK1 signaling is sufficient to transform EpH4 mammary epithelial cells and enhance invasiveness and metastasis in vivo without a requirement for EMT.


   ACKNOWLEDGMENTS
 
We thank Dr. Heidi Greulich for providing the Glu-Glu-tagged MEKDD cDNA construct, and Drs. Jeffrey Ecsedy, Stuart Martin, and Alan Ridgeway for critically reading the manuscript. Expert histopathologic analyses were provided by Dr. Roderick Bronson.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the Howard Hughes Medical Institute. Back

2 To whom requests for reprints should be addressed, at Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Warren Alpert Building, Room 539, 200 Longwood Avenue, Boston, MA 02115. Phone: (617) 432-7667; Fax: (617) 432-7944; E-mail: leder{at}rascal.med.harvard.edu Back

3 The abbreviations used are: MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; MAPK, mitogen-activated protein kinase; EMT, epithelial to mesenchymal transition; PI3k, phosphatidylinositol 3'-kinase; MEGM, mammary epithelial cell growth medium; CMV, cytomegalovirus; CREB, cyclic AMP-responsive element binding protein; MDCK, Madin-Darby canine kidney; TGF, transforming growth factor. Back

4 Internet address: http://www.clontech.com/atlas/genelists/index. Back

Received 3/19/02. Accepted 6/20/02.


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 DISCUSSION
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