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Cancer Research 67, 1618, February 15, 2007. doi: 10.1158/0008-5472.CAN-06-2913
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Prostate-Derived ETS Factor Is a Mediator of Metastatic Potential through the Inhibition of Migration and Invasion in Breast Cancer

David P. Turner1, Omar Moussa1, Moira Sauane2, Paul B. Fisher2 and Dennis K. Watson1

1 Department of Pathology and Laboratory Medicine, Department of Biochemistry and Molecular Biology and Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina and 2 Departments of Urology and Pathology, Herbert Irving Comprehensive Cancer Center, Columbia University, College of Physicians and Surgeons, New York, New York

Requests for reprints: Dennis K. Watson, Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425. Phone: 843-792-3962; Fax: 843-792-3940; E-mail: Watsondk{at}musc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell migration and invasion are critical events during the progression to metastasis. Without motile function, cancer cells are unable to leave the primary tumor site, invade through the basement membrane, and form secondary tumors. Expression of the epithelial-specific ETS factor prostate-derived ETS factor (PDEF) is reduced in human invasive breast tissue and lost in invasive breast cancer cell lines. Gain-of-function studies that examine different aspects of cell migration show that constitutive or inducible PDEF reexpression inhibits migration and invasion in multiple breast cancer cell lines, and loss-of-function studies show a stimulation of migration in noninvasive breast cancer cells. Furthermore, the introduction of PDEF into invasive breast cancer cells led to a remodeling of the actin cytoskeleton and altered focal adhesion localization and adherence levels. Cells expressing PDEF no longer form the defined morphologic polarity required for efficient, directional migration. Collectively, these data indicate that PDEF down-regulation in invasive breast cancer may promote actin-mediated cell migration through the extracellular matrix. [Cancer Res 2007;67(4):1618–25]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The WHO estimates that more than 1 million new cases of breast cancer will be diagnosed this year, resulting in the deaths of ~400,000 people.3 Metastasis accounts for the overwhelming majority of these deaths, as cells from the primary tumor migrate and invade into the bloodstream or lymphatic system and reestablish as secondary tumors (1, 2). To become invasive, cancer cells must dissociate from normal cell-to-cell contacts, reorganize their cytoskeleton, and transiently relocalize cell-matrix interactions to acquire the polarized morphology required for directed movement (3). These processes require extensive alterations in gene expression profiles, including the down-regulation of genes involved in cell anchorage and the up-regulation of genes involved in cell motility and matrix degradation (4).

The highly conserved ETS family of transcription factors functions in numerous biological processes, including cell proliferation, differentiation, apoptosis, angiogenesis, transformation, migration, and invasion (5–9). Abnormal ETS factor expression is associated with cancer progression in leukemias and solid tumors of the breast, prostate, colon, lung, pancreas, and thyroid. ETS factor overexpression has been observed in ductal breast carcinoma in situ and invasive breast carcinoma and is associated with the increased expression of several metastasis-associated genes. The expression of ETS1 or ETS2 stimulates breast cancer cell migration and invasion and is associated with the up-regulation of matrix-degrading proteins, such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (10–13).

Similarly, increased expression of the ETS family member PEA3 is also associated with increased migration and invasion in breast cancer cells (14) and is specifically associated with elevated HER-2/neu expression (15, 16), a marker for aggressiveness and lethality in breast cancer (17, 18).

Recently, our group has shown that ETS family members may also act as suppressors of metastasis. Expression of the epithelial-specific ETS factor prostate-derived ETS factor (PDEF) protein is reduced in invasive human breast (19) and prostate (20) cancer and lost in invasive breast cancer cell lines (19). Significantly, reexpression of PDEF in the invasive breast cancer cell line MDA MB 231 inhibits cell growth, migration, and invasion. These phenotypic changes are associated with the down-regulation of the metastasis activator uPA (19).

By examining the effect of PDEF expression in assays that investigate different aspects of cell migration, this study identifies for the first time that PDEF expression in invasive cancer alters the ability of motile cells to form the temporal structures required for efficient motility. Phenotypic alterations were found on both constitutive and inducible PDEF expression in multiple invasive breast cancer cell lines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Human breast epithelial cell lines were maintained at 37°C with 5% CO2 in medium supplemented with 10% fetal bovine serum and 100 units penicillin/streptomycin. BT549 cells were grown in RPMI 1640, whereas MDA MB 231, MDA MB 157, MDA MB 436, and MCF7 were grown in DMEM. MDA MB 231 and MDA MB 157 stable clones expressing doxycycline-inducible human PDEF were routinely grown in DMEM containing 150 µg/mL hygromycin and 200 µg/mL G418. All tissue culture reagents were purchased from Invitrogen (Carlsbad, CA).

Adenoviral infection. The construction of PDEF-expressing adenovirus has been previously described (19). Cells were infected in normal growth medium at 5 to 10 multiplicities of infection with either control virus expressing green fluorescent protein (Ad-GFP) or virus expressing PDEF/GFP from a bicistronic promoter (Ad-PDEF). Infected cells were then incubated as normal for 16 h. Under these conditions, >95% of the cells were infected as assessed by GFP expression.

Doxycycline-induced expression. MDA MB 231 and MDA MB 157 cells were transfected with 24 µg of EF1prTA, Tet-on construct (21) using LipofectAMINE 2000 according to the manufacturer's recommendations (Invitrogen). The cells were selected for neomycin resistance using 600 to 800 µg/mL of G418 (Invitrogen), and individual colonies were isolated after approximately 2 to 3 weeks of selection in the presence of 600 µg/mL G418. Each colony was tested for tetracycline responsiveness by transfecting 24 µg of pUHD 17-1 (Tet operator-regulated luciferase gene) in a 24-well format as described by Gopalkrishnan et al. (21). The BamHI PDEF insert prepared from PDEFpSG5neo (described in ref. 19) was cloned into the BglII site of the pUHD 10-3 vector modified to contain the hygromycin selection cassette. Stable clones were selected by growth in 150 µg/mL hygromycin, and inducible PDEF expression was determined following growth in doxycycline. Clones having no basal (minus doxycycline) expression were used for the studies described herein. Unless otherwise stated, PDEF expression was routinely induced at 70% to 80% confluence using 1 µg/mL doxycycline (Fisher Scientific, Rockville, MD) and incubated for 36 h before doing the experiments.

PDEF gene knockdown. PDEF was targeted using 21-mer small interfering RNA (siRNA) oligonucleotides (Dharmacon RNA Technologies, Chicago, IL) with the sequence 5'-AAGAACCGUCCCGCCAUGAAC-3'. MCF7 cells at 50% confluence were transfected with 10 nmol/L of PDEF-specific siRNA or 10 nmol/L scrambled control siRNA (Dharmacon RNA Technologies) using LipofectAMINE 2000 as per the manufacturer's instructions. Cells were incubated for a further 48 h before assays were done.

Western blot. Cells were lysed in radioimmunoprecipitation assay buffer containing protease inhibitors (Complete protease inhibitors, Roche, Nutley, NJ) and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO). Equal amounts of total protein were resolved by SDS-PAGE and subjected to Western blot analysis using enhanced chemiluminescence (Pierce, Rockford, IL). Human PDEF antibody was prepared as described previously (19). Other antibodies used were focal adhesion kinase (FAK) rabbit polyclonal (Cell Signaling Technology, Danvers, MA) and phosphorylated FAKtyr397 (pFAK) rabbit polyclonal and mouse monoclonal (BioSource, Camarillo, CA), actin rabbit polyclonal (Sigma-Aldrich), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rabbit polyclonal (Abcam, Cambridge, MA). For immunoprecipitation analysis of pFAK expression, 250 µg of total cell lysate were incubated overnight at 4°C with protein A dynabeads charged with FAK antibody and isolated as per the manufacturer's instructions (Invitrogen) before being resolved by SDS-PAGE and subjected to Western blot analysis using enhanced chemiluminescence.

Transwell migration and invasion assay. Treated or untreated control cells were seeded in triplicate into the upper chamber of a Transwell insert (BD Biosciences, San Jose, CA) in serum-free medium at a density of 50,000 per well. For migration assays, inserts were precoated with 5 µg/mL fibronectin (Fisher Scientific). Medium containing 10% serum was placed in the lower chamber to act as a chemoattractant, and cells were further incubated for 8 h. Nonmigratory cells were removed from the upper chamber by scraping, and the cells remaining on the lower surface of the insert were stained using Diff-Quick (Dade Behring, Inc., Newark, DE). Cells were quantified as the number of cells found in 10 random microscope fields in three independent inserts. Error bars represent the SD from three separate experiments. Invasion assays were done as for the migration assays described above, except inserts were precoated with the extracellular matrix (ECM) substitute Matrigel (BD Biosciences) and incubated over a 24-h period.

Migration track assay. Wells within a two-well chamber slide were precoated with 5 µg/mL fibronectin and then overlaid with a field of 1 µm in diameter carboxylate-modified polystyrene fluorescent microspheres (Invitrogen). Treated or untreated control cells were then seeded at low density (~4/mm2) in normal growth medium and incubated for a period of 24 h. The ability of the cells to create nonfluorescent tracks was then assessed by fluorescent microscopy and quantified using NIH image. Error bars represent the SD from three separate experiments. In a qualitative analysis, a mixed population of parental cells and cells infected with PDEF/GFP-expressing virus was seeded as above. GFP-expressing parental cells were visualized at 540 nm and PDEF/GFP-expressing cells as a merged image at 480/540 nm using an Olympus (Jamaica, NY) FluoView IX70 confocal microscope.

Wound assay. Cells were allowed to grow to 90% confluence in a six-well plate, and after either adenoviral-mediated or doxycycline-induced PDEF expression, cells were incubated for a further 16 and 36 h, respectively. After this time, the medium was replaced with serum-free medium and left overnight before scratching a wound down the center of the culture dish using a 200 µL pipette tip. Pictures of the denuded area were taken at time 0 and after 24 h using an Olympus FluoView IX70 confocal microscope. The denuded area was quantified at each time point using NIH image. Error bars represent the SD from three separate experiments.

Globular actin to filamentous actin ratio analysis. Treated or untreated control cells were grown to 70% to 80% confluence on a six-well plate. Cells were washed in PBS and lysed in 1% Triton X-100 in PBS for 2 min before collection. Cells were then centrifuged at high speed in a microcentrifuge, and the supernatant containing the globular actin (G-actin) was isolated. The remaining pellet containing the filamentous actin (F-actin) was then washed in PBS and resuspended in SDS loading buffer. Expression levels were then analyzed by Western blot and quantified using a Gel Doc system with the accompanying software [Kodak (New Haven, CT) ID 2.0.2]. Expression was quantified as the ratio of G-actin to F-actin, and error bars represent the SD from two separate experiments.

Morphology and cytoskeleton studies. Cell morphology analysis was done on glass chamber slides precoated with 5 µg/mL fibronectin. Treated or untreated control cells were then seeded at low density (~4/mm2) in normal growth medium and incubated for a period of 24 h. High-power images (x400 objective power) were taken at the indicated time points using an Olympus FluoView IX70 confocal microscope. For immunofluorescence studies, cells were seeded onto sterile coverslips (18 mm in diameter) coated with 5 µg/mL fibronectin and allowed to attach overnight. After either adenoviral-mediated or doxycycline-induced PDEF expression, cells were incubated for a further 16 and 36 h, respectively, before being fixed with 2% formaldehyde and permeabilized with 0.1% Triton X-100. Coverslips were blocked in 2% bovine serum albumin, and actin distribution was examined by phalloidin staining as per the manufacturer's instructions (Molecular Probes, Eugene, OR). Focal adhesion formation was examined in the same manner using pFAK polyclonal primary antibody (BioSource) and visualized using Alexa Fluor 488 goat anti-rabbit secondary antibody (Molecular Probes). Immunofluorescence was examined using an Olympus FluoView IX70 confocal microscope.

Adhesion assays. Fibronectin adhesion assays were done as described previously (22). Briefly, after incubation in the presence or absence of doxycycline, 40,000 cells were added to wells precoated with increasing concentrations of fibronectin (0–8 µg/mL). After 2 h of incubation, cells were extensively washed and fixed and the number of adherent cells was determined.

Statistical analysis. Statistical analyses were done using the Student's t test for paired data. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Constitutive PDEF expression inhibits chemokinetic migration. Compared with the endogenous levels of expression observed in MCF7 noninvasive breast cancer cells, infection with PDEF-expressing adenovirus produced high levels of protein in each of the four invasive breast cancer cell lines examined (MDA MB 231, MDA MB 157, BT549, and MDA MB 436; Fig. 1A ). Control cells infected with GFP-expressing virus had no detectable endogenous PDEF expression, confirming earlier reports (19). The effect of PDEF reexpression on chemokinetic migration (movement toward a stimulant) was examined in Transwell migration assays using serum as a chemoattractant. Compared with control cells, PDEF expression reduced the number of cells able to migrate across fibronectin-coated membranes by between 60% and 90% (Fig. 1B). Migration is a prerequisite for invasion through the basement membrane, and a similar inhibitory phenotype was observed when examining the invasive potential of cells expressing PDEF. Using membranes coated with the reconstituted basement membrane Matrigel, invasive potential was reduced by between 70% and 90% compared with control cells expressing GFP (Fig. 1C). We observed no evidence of cell death when doing the Transwell assays (data not shown), and therefore, apoptosis does not account for the inhibitory effects observed.


Figure 1
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  		Figure 1. PDEF expression inhibits chemokinetic migration and invasion in multiple invasive breast cancer–derived cell lines. A, Western blot analysis of PDEF expression in total cell lysates obtained from invasive breast cancer cell lines infected with GFP-expressing (G) or PDEF/GFP-expressing (P) adenovirus. Endogenous levels of PDEF expression are represented by total lysates from noninvasive MCF7 cells. B, quantification of cells migrating across fibronectin-coated Transwells. Columns, average number of cells migrating per 10 microscope fields. C, quantification of cells invading through Matrigel-coated Transwells. Columns, average number of cells migrating per 10 microscope fields. Each of the measured differences was statistically significant, with a P value of <0.05 by Student's t test.

 
Inducible PDEF expression inhibits chemokinetic migration. To look at the effect of PDEF at a more physiologic expression level, we generated stable MDA MB 231 and MDA MB 157 transfectants containing the EF1prTA, Tet-on and PDEF/UHD 10-3 vectors to allow doxycycline-inducible PDEF expression. Inducible expression of PDEF protein could be modulated in a dose-dependent (Fig. 2A, top ) and time-dependent (Fig. 2A, bottom) manner and produced levels of PDEF protein comparable with the endogenous level detected in MCF7 cells. At physiologic levels of expression, PDEF reduced chemokinetic cell migration by between 50% and 55% (Fig. 2B) and invasive potential by 55% to 60% (Fig. 2C). Inhibition of migration was time dependent, with the highest inhibition observed after 36 h in the presence of doxycycline (Fig. 2D). In reciprocal gene knockdown experiments done in the PDEF-expressing noninvasive breast cancer cell line MCF7, transfection with siRNA oligonucleotides directed against PDEF message successfully reduced PDEF protein levels by ~60% (Fig. 2E). Transwell migration assays show a significant stimulation of migration of PDEF siRNA-transfected cells (35%; Fig. 2E) compared with that of parental or MCF7 cells transfected with control siRNA oligonucleotides. Our previous work showed that PDEF reexpression induces a more rounded cell morphology in the MDA MB 231 cell line (19). This phenotypic change was also observed with the additional invasive cell lines used in this study and occurred when PDEF expression was either adenovirally or inducibly expressed (results not shown). Reciprocally, siRNA gene knockdown changed the morphology of MCF7 cells away from an epithelial to more elongated cell shape (Fig. 2F). Taken together, the Transwell data show that modulated PDEF expression significantly alters the migratory and invasive potential of breast cancer cell lines.


Figure 2
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  		Figure 2. PDEF expression and protein loss has reciprocal effects on chemokinetic migration. A, Western blot analysis of doxycycline (Dox)-induced PDEF expression in response to increasing dose (top) and incubation time (bottom) with doxycycline. Endogenous levels of PDEF expression are represented by total lysates from noninvasive MCF7 cells. B, quantification of cells migrating across fibronectin-coated Transwells in the presence or absence of doxycycline. Columns, average number of cells migrating per 10 microscope fields. C, quantification of cells invading through Matrigel-coated Transwells in the presence or absence of doxycycline. Columns, average number of cells migrating per 10 microscope fields. D, quantification of the timed response to treatment with doxycycline (1 µg/mL). Columns, average number of cells migrating per 10 microscope fields. Inset, representative stained Transwell membranes at each time point assayed. P value of <0.05 for each comparison. E, siRNA-mediated gene knockdown in MCF7 noninvasive breast cancer cells. Western blot analysis (inset) shows down-regulation of PDEF protein resulting from transfection with siRNA directed against PDEF (lane 3) compared with wild-type (lane 1) and control cells transfected with scrambled siRNA (lane 2). Quantification of the ability of the same cells to migrate across fibronectin-coated Transwells. Columns, total number of cells observed on the membrane. F, bright-field microscopy pictures of the morphology change observed on PDEF gene knockdown studies in noninvasive MCF7 breast cancer cells (Wild type) transiently transfected with scrambled control (Scrambled) and PDEF-directed (PDEF siRNA) siRNA oligonucleotides.

 
Modulated PDEF expression inhibits haptokinetic migration. The effect of PDEF reexpression on haptokinetic migration (movement across ECM) was first examined using the single-cell migration track assay. This assay examines the ability of individual cells to form migration tracks through a field of fluorescent microspheres. As the cells migrate, they engulf the spheres leaving a track of nonfluorescence, which can be readily quantified using NIH image (23). Differences in the track-forming abilities of a mixed population of PDEF-expressing and nonexpressing cells can be readily distinguished using strategic labeling of the populations (see Materials and Methods). Compared with control cells, PDEF expression significantly reduced the ability of cells to produce elongated migration tracks through the field of microspheres (Fig. 3A ). Interestingly, cells that express PDEF produced small rounded areas of sphere clearance (yellow fluorescence in Fig. 3A), which differ from the elongated tracks typically observed with PDEF minus parental cells (red fluorescence in Fig. 3A). In this single population haptokinetic assay across fibronectin, the average area migrated by an individual PDEF-expressing cell was reduced by between 50% and 70% compared with appropriate control cells (Fig. 3B). To assess the effect of PDEF expression on the haptokinetic migration of a population of cells, we did wound migration assays across fibronectin-coated plates (Fig. 3C and D). The ability of cells to migrate into the denuded area caused by a wound was significantly inhibited on PDEF expression when compared with appropriate control cells. Adenoviral-mediated expression inhibited migration by between 50% and 75%, and doxycycline-induced expression inhibited migration by between 55% and 60% (Fig. 3D). The inhibition of migration in both chemokinetic and haptokinetic migration assays indicates that PDEF reexpression in invasive breast cancer cells affects fundamental processes involved in cell motility.


Figure 3
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  		Figure 3. Modulated levels of PDEF expression inhibit haptokinetic migration. A, migration track assay done with a mixed population of BT549 parental cells and the same cells infected with PDEF/GFP-expressing virus. Green arrowheads, parental cells shown as areas of high red fluorescence; yellow arrowheads, PDEF-expressing cells shown as areas of high yellow fluorescence. B, quantification of the nonfluorescent tracks produced in migration track assays after adenoviral-mediated (first eight columns) and doxycycline-induced (last four columns) PDEF expression. Columns, average area migrated by individual cells either expressing or not expressing PDEF. C, representative immunofluorescent confocal image of the area migrated by MDA MB 157 cells infected with either GFP-expressing (Ad-GFP) or GFP/PDEF-expressing (Ad-PDEF) adenovirus. Pictures were taken at time 0 and after 24 h of incubation. D, quantification of the area migrated into the wound after adenoviral-mediated and doxycycline-induced PDEF expression. Columns, average area migrated by cells. Each of the measured differences obtained from the migration track and wound assays was statistically significant, with a P value of <0.05.

 
PDEF expression affects cell morphology through actin turnover. As noted above, close examination of the nonmigratory PDEF-expressing cells reveals a rounded area of cleared fluorescence rather than an elongated migration track seen in migratory control cells (Fig. 3A). This indicates that PDEF-expressing cells may still possess some motile function. Using bright-field microscopy, we show over time that, in a heterogeneous population, cells expressing PDEF adopt a rounded morphology and continually extend and contract cellular protrusions into the ECM (Fig. 4A ), resulting in the observed rounded areas of cleared fluorescence (Fig. 3A). GFP-expressing and uninduced control cells maintain the classic fibroblastic morphology of migrating cells, with cellular protrusions extending at the front and rear in the direction of movement.


Figure 4
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  		Figure 4. PDEF expression alters cell morphology and G-actin to F-actin ratios. A, representative bright-field confocal images of invasive breast cancer cells expressing or not expressing PDEF. PDEF expression was adenoviral mediated in MDA MB 231 cells (left) and doxycycline induced in stable MDA MB 157 cells (right). Photographs were taken at x400 objective power. B, quantification of the G-actin to F-actin ratio changes observed after adenoviral-mediated (first eight columns) and doxycycline-induced (last four columns) PDEF expression. Results are statistically significant, with a P value of <0.05. C, quantification and Western blot analysis of the G-actin to F-actin ratio changes induced by modulated PDEF expression in MDA MB 231 stable cells (dose response after 36 h of induction).

 
The extension and retraction of cellular protrusions is the result of a continuous cycle of monomeric G-actin polymerization and F-actin depolymerization (24, 25). A Western blot examination of the G-actin to F-actin ratio in PDEF-expressing cells compared with control cells reveals a distinct reduction in the levels of monomeric G-actin. Dependent on the cell line examined, the G-actin to F-actin ratio was reduced by between 50% and 70% (Fig. 4B) and can be modulated at different levels of PDEF expression (Fig. 4C). The decrease in the G-actin to F-actin ratio correlates with the increased number of cellular protrusions seen in PDEF-expressing cells.

PDEF expression alters F-actin distribution and polarity. The depletion of G-actin in PDEF-expressing cells may reflect an increase in the formation of F-actin structure within the cell as indicated by the increase in cellular protrusions. Using phalloidin staining and confocal microscopy, we observed a distinct pattern of F-actin structure formation in a heterogeneous population of PDEF-expressing cells when compared with control cells (Fig. 5 ). Cells not expressing PDEF displayed the classic actin structures associated with a migratory cell. These cells showed defined polarity in the direction of migration, with clearly discernible lamellipodia [flat protrusions of branched polymerized actin (26)] and filopodia [spike-shaped structures of parallel polymerized actin bundles (27, 28)] at the leading edge, and possessed a distinct trailing edge (Fig. 5, columns 1 and 2). PDEF-expressing cells, however, lacked any defined polarity with no obvious leading edge and displayed an increased number of F-actin structure, including both lamellipodia and filopodia, which stretched out into the ECM with no defined directional polarity (Fig. 5, columns 3 and 4).


Figure 5
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  		Figure 5. PDEF expression alters actin distribution. Phalloidin staining of F-actin distribution and structure after adenoviral-mediated (rows 1–4) and doxycycline-induced (rows 5 and 6) PDEF expression. Group cell photographs were taken at x100 objective power, and single cell photographs were taken at x400 objective power.

 
PDEF expression alters focal adhesion localization and cell adhesion. Cell polarity during migration is thought to be regulated by a cycle of focal adhesion assembly and disassembly at the leading and trailing edge of migrating cells (29). The lack of morphologic polarity in PDEF-expressing cells suggests that PDEF may affect adhesion complex formation. We therefore examined the effect of PDEF expression on focal adhesion formation using immunofluorescence microscopy with pFAK antibody. FAK does protein-protein adapter functions critical to the formation of focal adhesions and mediates adhesion and growth factor–dependent signals into the cell (30). Nonmigratory PDEF-expressing cells displayed distinct changes in focal adhesion localization when compared with migratory control cells. Focal adhesions present in control cells had defined polarity in the direction of migration and localized predominantly at the leading edge lamellipodia and within the trailing edge (Fig. 6A ). Adhesion complexes were observed mainly at the cell periphery, and those present on the basolateral surface had defined directionality with the leading edge. In cells expressing PDEF, however, focal adhesions lacked defined polarity and were observed all around the basolateral surface of the cell (Fig. 6A). An examination of protein levels revealed a small but reproducible reduction in FAK phosphorylation in cells expressing PDEF (Fig. 6B). Although this was consistently observed in both stable cell lines, the reduced levels were only statistically significant in the higher PDEF-expressing MDA MB 231 stable cell line (P = 0.007), suggesting that the levels of PDEF expression correlate with pFAK localization. Focal adhesion formation and its interaction with the ECM plays a crucial role in the dynamics of cell migration. Reduced adhesion decreases cell traction and increased adhesion renders cells unable to move (31). When PDEF was inducibly expressed in MDA MB 231 and MDA MB 157 cells, the ability of the cells to adhere to fibronectin was significantly reduced when compared with uninduced control cells (Fig. 6C). The combined data from the examination of F-actin distribution, focal adhesion localization, and cell adhesion indicate that PDEF-mediated inhibition of migration and invasion may occur through pathways mediating cytoskeleton reorganization and ECM interactions.


Figure 6
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  		Figure 6. PDEF expression results in altered focal adhesion localization and cell adhesion. A, immunofluorescent staining of adhesion complex localization in two stable PDEF-inducible cell lines after treatment with (Plus PDEF) or without (Minus PDEF) doxycycline. Adhesion complex localization was visualized using immunofluorescent staining of pFAK (480 nm) and F-actin (540 nm). Colocalization as a merged image at 480/540 nm. B, representative Western blot analysis of pFAK expression. Total cell lysates obtained from stable PDEF-inducible cells in the presence and absence of doxycycline were immunoprecipitated with antibody against total FAK and visualized using Western blot analysis with pFAK-specific antibody. Protein levels of PDEF and GAPDH as expression and loading controls, respectively. C, quantitative assessment of the ability of cells to adhere to increasing concentrations of fibronectin in the presence or absence of doxycycline. P ≥ 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The regulation of cell migration and invasion is critical to the progression of cancer. This study shows for the first time that PDEF-mediated inhibition of migration and invasion occurs at physiologic levels of expression in multiple invasive breast cancer cell lines and that these inhibitions correlate with distinct changes in cytoskeleton morphology. Significantly, our data uniquely identify that PDEF expression disrupts the ability of invasive cells to form the temporal structures required for polarized movement through the ECM. PDEF protein loss in invasive cells may therefore be a critical factor in the progression of cancer by allowing cells to form the defined morphologic polarity required for directed migration. Our earlier work showed that invasive breast cancer cell lines do not have detectable levels of PDEF protein and that its reexpression in the invasive MDA MB 231 breast cancer cell line inhibits cell growth, migration, and invasion (19). The data in this article show that these effects are shown using several methods of analysis, across multiple invasive breast cancer cell lines, using adenoviral overexpression and inducible expression systems. Our data also show that PDEF gene knockdown in the noninvasive MCF7 breast cancer cell line stimulates migration (Figs. 2E and 5B). Interestingly, PDEF gene silencing in MCF7 cells has recently been shown to accelerate xenograft tumor formation in mice (32). Together, our gain-of-function and loss-of-function studies provide strong evidence for a suppressive role for PDEF in migration and invasion. However, a recent retroviral-based migration screen has identified PDEF as a possible stimulator of cell migration in MCF7, LoVo, and other cancer cell lines (33). Increased PDEF expression, in conjunction with either HER-2/neu or colony-stimulating factor receptor 1 (CSFR1) up-regulation, also was found to increase migration and cell transformation in soft agar studies. However, no demonstration of PDEF expression level was provided for the systems examined. Of note is the finding that increased PDEF expression alone only produced a limited stimulation on cell migration and had no effect on cell transformation. In our Transwell migration assays, increased expression of PDEF produced no significant change in the migratory ability of MCF7 cells (which already have endogenous expression of PDEF; results not shown) and expression inhibited migration in LoVo colon cancer cells (which do not express endogenous PDEF protein).4 One difference in methodology between the two studies is that our Transwell assays were done over fibronectin-coated polycarbonate membranes rather than uncoated membranes. It remains to be determined whether HER-2/neu or CSFR1 expression provides a unique cell context that may account for the stimulation of migration observed following PDEF coexpression. In the same publication (33), the authors show that PDEF mRNA is overexpressed in human tumor samples throughout tumor progression; however, no demonstration of increased PDEF protein levels was provided. Our work, and the work of other laboratories, has shown that although PDEF mRNA can be found to be overexpressed in tumors and tumor cell lines (34–36), PDEF protein levels are not always correlated with mRNA levels, suggesting posttranscriptional down-regulation. To date, this has been shown in breast (19, 32), prostate (20, 34), and colon cancer.4

To become motile, cells must establish and maintain a defined polarity in the direction of movement. This is typified by a morphologic polarization consisting of a dominant lamellipodia at the leading edge and an extended trailing edge at the rear (37–39). At the onset of motility, several lamellipodia and filopodia protrude from the cell until a dominant lamellipodia is established (40). Newly formed focal adhesions then adhere the leading lamellipodia to the ECM (31, 41, 42). PDEF expression in invasive cells leads to an increased number of cellular protrusions (Fig. 4A) as well as F-actin structure (Figs. 4B and 5A) and relocalizes weakened focal adhesions to the basolateral surface (Fig. 6). This indicates that PDEF-expressing cells may fail to establish the leading lamellipodia required for polarized migration, which in turn may be due to an inability to establish newly formed focal adhesion complexes at this site.

In conclusion, a defining characteristic of a metastatic cancer cell lies in its ability to migrate. Expression of the ETS transcription factor PDEF in invasive breast cancer alters the ability of cancer cells to form the temporal structures and defined morphologic polarity required for efficient migration and invasion. Using gene expression profiles and subsequent gene ontology analysis, our future studies will attempt to identify the specific cell-cell and cell-matrix interactions involved in PDEF-mediated inhibition of cell migration. Elucidation of the mechanisms involved in cancer cell migration may facilitate the design of novel molecularly based diagnostic and therapeutic approaches to cancer treatment.


   Acknowledgments
 
Grant support: NIH grants PO1-CA78582 (D.K. Watson) and RO1 CA35675 (P.B. Fisher) and DAMD W81XWH-04-1-0433 (M. Sauane and P.B. Fisher).

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.

We thank Drs. Victoria J. Findlay and Patricia M. Watson for critical review of this manuscript, Fan Fan for technical assistance, and Dr. Ed Krug (Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC) for his helpful discussion of actin dynamics.


   Footnotes
 
3 http://www.who.int/whosis/en Back

4 O. Moussa et al., in preparation. Back

Received 8/ 7/06. Revised 11/10/06. Accepted 12/22/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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