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Carcinogenesis Advance Access originally published online on February 1, 2007
Carcinogenesis 2007 28(6):1153-1162; doi:10.1093/carcin/bgm015
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

BMP4 induces EMT and Rho GTPase activation in human ovarian cancer cells

Brigitte L. Thériault, Trevor G. Shepherd, Michelle L. Mujoomdar and Mark W. Nachtigal*

Department of Pharmacology, Faculty of Medicine, Dalhousie University, Sir Charles Tupper Medical Building, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5

* To whom correspondence should be addressed. Tel: +902 494 6348; Fax: +902 494 1388; Email: mark.nachtigal{at}dal.ca


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
We identified previously an autocrine bone morphogenetic protein-4 (BMP4) signalling pathway in primary human normal ovarian surface epithelial (OSE) and epithelial ovarian cancer (OvCa) cells. Herein we show that treatment of OvCa cells with BMP4 produced morphological alterations and increased cellular adhesion, motility and invasion. The BMP4 inhibitor noggin blocked the BMP4-induced phenotype, and decreased autocrine BMP4-mediated OvCa cell motility and adherence. In response to exogenous BMP4, the epithelial–mesenchymal transition (EMT) markers Snail and Slug mRNA and protein were up-regulated, E-cadherin mRNA and protein were down-regulated and the network of alpha smooth muscle actin changed to resemble a mesenchymal cell. We also observed changes in the level of activated Rho GTPases in OvCa cells treated with BMP4, strongly suggesting that the changes in morphology, adhesion, motility and invasion are probably mediated through the activation of these molecules. Strikingly, treatment of normal OSE cells with BMP4 or noggin failed to alter cell motility, providing evidence that OSE and OvCa cells possess a distinct capability to respond to BMP4. Overall, our studies suggest a link between autocrine BMP signalling mediated through the Rho GTPase family and Snail- and Slug-induced EMT that may collectively contribute to aggressive OvCa behaviour.

Abbreviations: BMP, bone morphogenetic protein; BSA, bovine serum albumin; EMT, epithelial–mesenchymal transition; FAP, focal adhesion protein; FBS, fetal bovine serum; LIMK1, LIM Kinase 1; NT, non-transduced; OSE, ovarian surface epithelial; OvCa, ovarian cancer; PBS, phosphate-buffered saline; QPCR, quantitative polymerase chain reaction; RT, room temperature


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
Bone morphogenetic proteins (BMPs) are expressed in many adult tissues and play important functions in adult tissue formation, maintenance, remodelling and repair (1–3). BMP dimers transduce their signals through a heterotetrameric complex consisting of type I (ALK2, 3, 6) and type II (BMPRII) serine/threonine kinase transmembrane receptors. Subsequent intracellular signalling is mediated through activation of Smads 1, 5 and 8 interacting with the common Smad4, and upon nuclear translocation modifies target gene expression (4). Many BMPs are expressed in the ovary (BMP2, 3, 3b, 4, 6, 7, 15) and contribute to the processes of follicle maturation and steroidogenesis (5,6). BMP4 in particular is expressed in a spatially and temporally distinct manner in the ovarian surface epithelial (OSE) layer of cells immediately following ovulation, and based on this expression profile it was suggested that BMP4 signalling may participate in OSE wound repair following ovulation (7).

Current theories suggest that most ovarian cancers (OvCas) arise from the OSE (8). The OSE forms a monolayer of flat, cuboidal or low columnar epithelial cells on the ovarian surface separated from the underlying stroma by a basement membrane. The OSE functions to transport molecules between the ovary and the peritoneal cavity, and participates in ovulation through the local production of proteases at the site of follicle rupture (8). In response to ovulation, OSE cells divide and migrate to seal the ovulatory wound. It is hypothesized that repeated cycles of wounding, proliferation and repair may contribute to accumulation of genetic damage and the development of OvCa (9). In the rat, it has been demonstrated that coincident with ovulation, BMP4 is up-regulated in OSE adjacent to the site of ovulation (7). These correlative data suggest that BMP4 may participate in OSE repair through promoting cellular division or migration. We have demonstrated that normal human OSE and epithelial OvCa cells possess an autocrine BMP signalling pathway (10). BMP signalling can produce alterations in the behaviour of a diverse array of cancer cells, ranging from growth inhibition and apoptosis (11–16) to influencing metastatic potential (17–20). This report identifies differences in the BMP4 signalling response between normal OSE and OvCa cells.

We determined previously that target gene expression in response to BMP4 stimulation is more pronounced in OvCa cells versus normal OSE cells, and treatment with exogenous BMP4 can produce a cell-spreading phenotype (10). While BMP4 does not affect the growth rate of OvCa cells, BMP4-induced cell spreading resulted in decreased saturation density. Herein we examine the morphological and phenotypic consequences of BMP signalling in both primary human normal OSE and OvCa cells. BMP4-treated OvCa cells show changes in their behaviour including increased motility, adhesion and invasion, and undergo a remodelling event resembling an epithelial–mesenchymal transition (EMT). EMT is associated with enhanced cellular motility and invasiveness (21,22), which contributes to cancer cell transition through more aggressive stages. In contrast, normal OSE cells do not respond to BMP4 by altering cell motility, identifying an important difference between the response of normal OSE and OvCa cells to exogenous BMP4. We determined that the BMP inhibitor noggin could abrogate autocrine BMP4-induced activities, highlighting the contribution of BMP4 signalling to aggressive cellular behaviours. BMP4-induced EMT of OvCa cells correlates with re-organization of the actin cytoskeleton and activation of the Rho GTPases. Collectively, these results indicate that BMP4 signalling may contribute to OvCa progression.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary data
 References
 
Culture of primary human OSE or OvCa cells
Institutional approval for research with human materials was received prior to the initiation of these studies (QEII Health Sciences Centre, Research Ethics Committee, #QE-RS-99-016; IWK/Grace Hospital Research Ethics). Primary human OSE cells were isolated and grown as described previously (23). Cells were treated with recombinant human BMP4 (R&D Systems, Minneapolis, MN, USA) at a concentration of 10 ng/ml (278 pM) or noggin/Fc (R&D Systems) at a concentration of 100 ng/ml. BMP4 and noggin were reconstituted in 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) (vehicle). Primary human OvCa cells were isolated from ascites fluid obtained from patients with stage III or IV OvCa, and grown as described previously (23). All experiments with OvCa cells were performed between passages 2 and 6.

Adenoviral constructs and cell transduction
The constitutively active BMP type IA receptor cDNA (ALK3QD; kind gift from Dr. L.Attisano, University of Toronto) was used to generate adenovirus-expressing ALK3QD (Ad-ALK3QD) using the AdEasy Vector System (Qbiogene, Irvine, CA, USA) according to the manufacturer's protocol. Ad-ALK3QD particles were purified using the BD Adeno-X Virus Purification kit (BD Biosciences Clontech, Palo Alto, CA, USA). Ad-green fluorescent protein (GFP) was a kind gift from Dr B.C.Vanderhyden (Ottawa Health Research Institute). Primary OvCa cells were transduced with a multiplicity of infection of 200 of Ad-ALK3QD or Ad-GFP in medium containing 10% fetal bovine serum (FBS) for 90 min with occasional agitation. After transduction, complete growth medium was replenished. ALK3QD is tagged with a hemagglutinin (HA) epitope; anti-HA was used to detect expression by western analysis (Figure 1C, XIII).


Figure 1
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  		Fig. 1. BMP4 signalling induces cytoskeletal re-organization and alters morphology of normal OSE and OvCa cells. (A) Normal OSE cells (I–VI) treated with vehicle or BMP4 (10 ng/ml). (I and IV) Cells were visualized by phase-contrast microscopy. Magnification x200. (II and V) OSE cells stained with Alexa 488-conjugated phalloidin to reveal filamentous actin stress fibres (green), visualized by fluorescence microscopy. (III and VI) OSE cells stained with anti-vinculin plus Alexa 488-conjugated secondary antibodies (green), visualized through fluorescence microscopy. (II, III, V and VI) Samples were co-stained with Hoechst 33258 to reveal nuclei (blue). (B) OvCa cells treated with vehicle or BMP4 (10 ng/ml). (I and V) Cells were visualized by phase-contrast microscopy. Magnification x200. (II and VI) OvCa cells stained with Alexa 488-conjugated phalloidin to reveal filamentous actin stress fibres (green), visualized by fluorescence microscopy. (III and VII) OvCa cells stained with anti-vinculin plus Alexa 488-conjugated secondary antibodies (green), visualized through fluorescence microscopy. (IV and VIII) OvCa cells co-stained with rhodamine-conjugated phalloidin (red) and anti-vinculin plus Alexa 488-conjugated secondary antibodies (green), visualized through confocal microscopy. Magnification x630. Co-localized protein appears yellow. Similar results for experiments in (A) and (B) were observed using primary cells from 9 normal OSE and 18 OvCa samples, respectively. Bar, 50 µm. (C) The morphology of NT OvCa cells (I–IV), OvCa cells transduced with Ad-ALK3QD (V–VIII) or transduced with Ad-GFP (IX–XII) was visualized over 5 days by phase-contrast microscopy. (IV, VIII and XII) On day 5, F-actin was visualized using rhodamine–phalloidin staining (red) and fluorescence microscopy; samples were co-stained with Hoechst 33258 to reveal nuclei (blue). Similar results were obtained using primary cells from five OvCa samples. (XIII) Increasing multiplicity of infection of Ad-ALK3QD was used to transduce primary OvCa cells. ALK3QD was detected by western analysis; actin was examined to assess protein loading. Bar, 50 µm.

 
Fluorescence and confocal microscopy
Primary OvCa cells were grown to 70–90% confluence on 18 mm diameter round glass cover slips placed in 22 mm diameter culture dishes. After treatment, growth medium was removed and cells were washed with PBS, fixed with 4% paraformaldehyde in PBS for 10 min at room temperature (RT) and permeabilized with 0.3% Triton X-100 (Thermo Fisher Scientific, Ottawa, ON, Canada) for 10 min at RT. Vinculin staining was done through indirect immunofluorescence employing a 1:100 dilution of a mouse anti-human vinculin primary antibody (Sigma Aldrich Canada, Mississauga, ON, Canada) incubated with 10% donkey serum in PBS overnight at 4°C. Secondary antibody dilutions [1:1000 goat anti-mouse Alexa 488 (Invitrogen Molecular Probes, Burlington, ON, Canada)] were incubated with 0.05% Tween 20 (Fisher) and 0.1% BSA (Fisher) in PBS for 2 h at 37°C. F-actin stress fibres were visualized by staining fixed and permeabilized OvCa cells with a 1:1000 dilution of either Alexa 488-conjugated phalloidin or rhodamine-conjugated phalloidin (Molecular Probes) in 0.05% Tween 20 and 0.1% BSA for 1 h at 37°C, followed by incubation with Hoechst 33258 stain (1:1000 in PBS) for 15 min at 4°C to identify nuclei. Fluorescence images were obtained using a Zeiss Axiovert 200 inverted microscope with Zeiss Axiocam HRc digital camera at RT; x20 objective has a numerical aperture of 0.5 and x40 objective has a numerical aperture of 0.6. Confocal images were taken using a Zeiss LSM 510 microscope equipped with argon (458/488 nm) and helium (548/633 nm) lasers, capturing 0.1 µm sections throughout the cell at RT; x63 objective has a numerical aperture of 1.4. Digital images were processed using Adobe Photoshop 7.0.

RNA isolation and quantitative polymerase chain reaction
Total cytoplasmic RNA was isolated from cultured OvCa cells as described previously (10). cDNA was generated from 2 µg of total RNA using Stratascript reverse transcriptase (Stratagene, La Jolla, CA, USA). Quantitative polymerase chain reaction (QPCR) was performed using the Brilliant SYBR Green® QPCR Master Mix and the Mx3000P QPCR machine and analysis software (Stratagene). Expression of integrin receptors, extracellular matrix (ECM) proteins, focal adhesion proteins (FAPs), filamin, and glyceraldehyde-3-phosphate dehydrogenase was assessed by QPCR (35 cycles: 30 s at 95°C, 30 s at 59°C, 30 s at 72°C) using primers specific for each human cDNA sequence. Fold difference in mRNA expression before and after BMP4 treatment was calculated using the normalized Ct value obtained from the Mx3000P analysis software as reported previously (24). The identity of the PCR amplicons was verified by sequencing (DNA Sequencing Facility, Dalhousie University). Data were obtained from a minimum of three independent experiments conducted in duplicate for each patient sample. A total of 11 patient samples were analysed.

Wounding assay
Normal OSE or primary OvCa cells were grown to 100% confluence on 18 mm diameter culture dishes or 25 mm square glass cover slips photo etched with a lettered and numbered grid (Bellco Glass Inc., Vineland, NJ, USA) in 35 mm diameter culture dishes, respectively. After treatment, growth medium was removed, cells were washed with PBS and a wound was produced (~1 mm) (25). Cells were rinsed with PBS to remove non-adherent cells, and fresh growth media was added. Digital images were taken at the same grid coordinates at time points up to 12 h with a Nikon TMS phase-contrast microscope equipped with a Nikon Coolpix 4500 digital camera. The percentage of total area covered by the cells in each image was calculated using the National Institutes of Health image analysis software program Image J.

Adhesion assay
Primary OvCa cells were cultured to 70–80% confluence and adhesion assays performed as described previously (26). Where indicated, wells were previously coated with 500 ng/cm2 of ECM proteins (Sigma), allowed to air dry and blocked with 1% BSA in PBS (vehicle) for 1 h at 37°C.

Invasion assays
Primary OvCa cells were cultured in 25 cm2 flasks and pre-treated with BMP4 (10 ng/ml) or transduced for 48 h with either Ad-ALK3QD or Ad-GFP. Invasion assays were performed overnight as described previously (26) where a collagen I (BD Biosciences) gel was pre-coated onto Transwell culture inserts. OvCa cells that had invaded through the Transwell culture insert filter were counted by fixing the cells with ethanol and staining with Mayer's haematoxylin. Results are expressed as the mean number of cells per six high-power fields of view that had invaded through the collagen gel and the culture insert filter.

Re-plating and proliferation assay
OvCa cells were treated with vehicle or BMP4 (10 ng/ml) for 48 h, trypsinized and re-plated in the presence or absence of BMP4. Cell morphology was visualized by phase-contrast microscopy. Magnification x200. OvCa cell proliferation was determined by counting the cell number using a haemacytometer throughout the growth period.

Rho GTPase activation assays and western analysis
Activation of Rac1, Rho and Cdc42 GTPases was assayed using the Pierce GTPase Activation kits as described previously (27). Briefly, primary OvCa cells were grown to 70–80% confluence, incubated in low-serum conditions (0.2% FBS in growth medium) overnight and treated for up to 48 h with 10 ng/ml BMP4 (R&D Systems). After treatment, GTPase activation was assessed as per manufacturer's instructions. Samples were run on a 15% polyacrylamide gel. Rac1 (1:1000), Rho (1:500; detects Rho A, B and C) and Cdc42 (1:500) were detected with monoclonal antibodies (Pierce Biotechnology, Rockford, IL, USA). A 1:5000 dilution of sheep anti-mouse secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA, USA) was used to detect GTPase expression using enhanced chemiluminescence and STORM scanner digital imagery (Amersham, GE Healthcare, Baie d'Urfe, QC, Canada). Snail and Slug (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and E-cadherin (1:250, Cell Signaling Technologies, Danvers, MA, USA) proteins were detected by enhanced chemiluminescence using a 1:5000 dilution of sheep anti-rabbit secondary antibody conjugated to horseradish peroxidase (Chemicon).

Statistical analyses
For all experiments where P values are expressed, a paired Student's t-test was performed, where significance was set at P < 0.05. Unless otherwise stated, the P values represent data obtained from three independent experiments done in triplicate.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary data
 References
 
BMP4 induces cytoskeletal re-organization and alters morphology of normal OSE and OvCa cells
To assess the change in normal OSE and OvCa morphology in response to BMP4, primary human cells were obtained from normal ovarian tissue biopsies or from ascites fluid samples originating from chemotherapy naive OvCa patients. While the majority of OvCas are of the serous subtype, cells were obtained from patients diagnosed with serous, mucinous and endometrioid subtypes and similar results were observed with cells from these subtypes. OSE or OvCa cells were treated with 10 ng/ml (278 pM) recombinant human BMP4 for up to 7 days. BMP4 was selected for these studies because we had shown previously that BMP4 was expressed in human OSE and primary OvCa cells, whereas other BMP family members were rarely (BMP2) or not (BMP7) detected, and exogenous BMP4 produced changes in BMP target gene expression in these cells (10). Phase-contrast images show a distinct increase in cell size in response to BMP4 treatment in OSE and OvCa cells (Figure 1A and B). On average, OvCa cells increase their cell area 2.8-fold in response to BMP4 treatment (P < 0.0001; OSE, 10 samples; OvCa, 18 samples). We had reported previously that BMP4 treatment did not change the general cytoskeletal architecture of primary OvCa cells when assessed using anti-actin by indirect immunofluorescence (10). To refine these previous analyses, we switched our technique to use phalloidin, which is designed to specifically detect filamentous actin, in combination with confocal microscopy, as opposed to using indirect immunofluorescence and the anti-actin antibody that detects both globular and filamentous actin. The experiments conducted using phalloidin have allowed us to more clearly visualize the alterations in the actin cytoskeleton. Herein we show a re-organization of the actin cytoskeleton from the predominance of cortical actin into actin stress fibres throughout the cell in response to BMP4 treatment. In the previous experiments, our analyses using anti-actin likely masked this effect because of the high level of globular actin present in the cells and the high level of background staining. Re-organization of filamentous actin is characteristic of a cell-spreading response (28). Cell spreading requires changes in adhesion to the ECM (29–31). Vinculin is a FAP that nucleates many molecular components, including F-actin, which is required for cellular motility and adhesion. BMP4 treatment induced a re-localization of vinculin to membrane ruffles and co-localized with the termini of F-actin stress fibres (Figure 1B, VIII). Co-localization of vinculin and F-actin does not occur in the vehicle-treated cells (Figure 1B, IV). F-actin and vinculin co-localization suggests that these molecules form a complex that participates in cellular remodelling and enhanced adhesion and motility.

To further evaluate the contribution of BMP signalling to morphological changes, OvCa cells were transduced with an adenovirus expressing a constitutively activated BMP type IA receptor, ALK3QD. ALK3 was selected for these studies because we had determined that the BMP type IB receptor (ALK6), which is also capable of transmitting BMP4 signals, is rarely expressed in OSE or OvCa cells (10). These analyses were conducted only in OvCa cells due to the limited availability of normal OSE cells; OSE cells were used for assays requiring fewer cells. Compared with non-transduced (NT; Figure 1C, I–IV) OvCa cells, Ad-ALK3QD transduced cells showed consistent changes in morphology and cell area after transduction (5-fold increase in area, P < 0.0004; Figure 1C, V–VIII). To control for adenoviral transduction and protein over-expression, OvCa cells were transduced with Ad-GFP (Figure 1C, IX–XII); these cells did not exhibit morphological alterations compared with NT cells. ALK3QD expression was detected by western analysis (Figure 1C, XIII). For transduction experiments, a multiplicity of infection of 200 was selected because it produces ~100% OvCa cell transduction efficiency.

BMP4 increases mRNA expression of integrins, FAPs and ECM proteins
To assess the mRNA expression of a subset of molecules that control cell morphology, adhesion and migration, quantitative RT–PCR (QPCR) analysis was performed on total RNA isolated from cultures of OvCa cells treated from 1 to 7 days with vehicle or 10 ng/ml BMP4. An increase in the mRNA of many integrin subunits, FAPs and ECM proteins in response to BMP4 treatment was observed at different time points (supplementary Table SI is available at Carcinogenesis online). These data show that BMP4 can induce alterations in the expression of molecules that participate in cellular interaction with the extracellular environment.

BMP4 induces an EMT response in OvCa cells
The morphological response of primary OvCa cells to BMP4 treatment resembles an EMT. In response to BMP4 treatment, the staining pattern for alpha smooth muscle actin (SMA) was altered and persisted for up to 8 days (Figure 2A). Smooth muscle actin distribution was similar to the staining pattern observed in mesenchymal cell types (32). These cells continued to proliferate after undergoing morphological alterations, were efficiently lifted and re-plated (~83% average re-plating efficiency for vehicle- or BMP4-treated cells) and maintained their mesenchymal morphology after re-plating (supplementary Figure S1 is available at Carcinogenesis online). Additionally, mRNA levels for the EMT markers Snail and Slug increased an average of 2-fold as early as 1 h after BMP4 treatment (Figure 2B). Changes in mRNA preceded increased Snail and Slug protein levels (Figure 2C). In contrast, mRNA for the epithelial marker E-cadherin was not altered initially in the presence of BMP4; however, mRNA and protein levels decreased (~50% compared with untreated cells) by 48 h and persisted at lower levels until the experiment was terminated (Figure 2B and C). These results are consistent with transcriptional repression of E-cadherin by Snail and Slug (33). Taken together, these data strongly suggest that exposure to exogenous BMP4 can induce an EMT in primary OvCa cells.


Figure 2
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  		Fig. 2. BMP4 induces an EMT response in OvCa cells. (A) OvCa cells were treated with vehicle (upper panels) or BMP4 (bottom panels) for up to 8 days. Expression of alpha smooth muscle actin was assessed by immunofluorescence (green) and Hoechst 33258 to reveal nuclei (blue). Bar, 50 µm. Similar results were obtained using primary cells from two OvCa samples in triplicate. (B) Mean data from five OvCa cell samples showing expression of various EMT markers (Snail, Slug and E-cadherin) assessed using QPCR over a 6 day period in cells treated with BMP4. Data are shown as fold change in expression between BMP4-treated relative to vehicle-treated cells. Bars represent standard deviation from three independent experiments conducted in duplicate. *P < 0.05. (C) Mean data from three OvCa cell samples showing expression of Snail, Slug or E-cadherin protein assessed over a 6 day period in cells treated with BMP4. Data are shown as fold change in expression between BMP4-treated relative to vehicle-treated cells. Bars represent standard deviation of protein expression from the three patient samples. *P < 0.05.

 
OvCa cell adhesion (reattachment) is increased in response to BMP4
To functionally assess the ability of OvCa cells to interact with various ECM substrates, OvCa cells (pre-treated with vehicle or BMP4 for 48 h) were plated on culture dishes coated with control (1% BSA in PBS) or various ECM substrates and adhesion assays were performed. BMP4 induced a significant increase in cell adhesion, and this was the case for all the ECM coatings tested (Figure 3). OvCa cells also showed selective interaction with different ECM substrates, as indicated by the highest adhesion on fibronectin and the lowest adhesion on laminin (Figure 3B, C and D). Additionally, while vehicle-treated OvCa cells appear to have similar morphologies when cultured on glass, collagen I, collagen IV or fibronectin, these cells have an altered morphology when cultured on laminin (supplementary Figure S2 is available at Carcinogenesis online). These results suggest that OvCa cells do not adhere or interact with all ECM substrates in the same manner. In response to BMP4, OvCa cells display a cell-spreading phenotype when cultured upon all ECM substrates including laminin. Transduction of OvCa cell samples with Ad-ALK3QD also significantly increased cell adherence to all coatings tested (Figure 3E and supplementary Figure S3 is available at Carcinogenesis online). To determine if inhibition of BMP4 signalling would affect cell adherence, cells were also co-treated with either BMP4 plus the BMP inhibitor noggin/Fc [100 ng/ml; (34)] or noggin/Fc alone for 48 h prior to assessing reattachment. Treatment with BMP4 plus noggin/Fc or noggin/Fc alone significantly reduced cell reattachment to below control (vehicle) levels on coating control, collagen I and collagen IV, suggesting that noggin/Fc is inhibiting autocrine BMP signalling activity that may regulate basal adhesive protein levels (Figure 3E and supplementary Figure S3 is available at Carcinogenesis online).


Figure 3
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  		Fig. 3. OvCa cell adhesion (reattachment) is increased in response to BMP4. Representative data from one patient sample showing OvCa cells that were pre-treated with BMP4 for 48 h, radiolabelled with 3H-amino acids, lifted and re-plated onto (A) coating control (1% BSA in PBS), (B) fibronectin or (C) laminin. The percentage of the cells that adhered was assessed up to 240 min post-plating. (D) The percentage of cells that adhered at 240 min is shown for all ECM substrates tested. (E) OvCa cells were pre-treated with vehicle, BMP4, Ad-ALK3QD, BMP4 and noggin/Fc or noggin/Fc for 48 h, radiolabeled with 3H-amino acids, lifted and re-plated onto collagen IV. The percentage of the cells that adhered was assessed at various times up to 240 min post-plating. Bars represent standard deviation from three independent experiments conducted in triplicate. *P < 0.05. Similar results were obtained using primary cells from three OvCa samples.

 
BMP4 increases OvCa cell motility but not normal OSE cell motility
A wounding assay was used to examine cell motility of OvCa cells in response to BMP4. OvCa cells were treated with vehicle or BMP4 for 48 h prior to producing a 1 mm wound. The ability of these cells to close the wound was then assessed over time. Over a period of 12 h, the BMP4-treated cells migrated to cover a greater surface area of the wound as compared with vehicle-treated cells (Figure 4). A significant increase in the percentage of the total area covered by cells that were subjected to BMP4 treatment was apparent 6 h after producing the wound (Figure 4B). At 12 h, the average increase in motility for BMP4-treated samples is a 2-fold increase (P < 0.0005). Transduction of OvCa cells with Ad-ALK3QD also showed a significant increase in cell motility (Figure 4C and D). In addition, noggin/Fc abrogated cell motility induced by BMP4 treatment, and treatment of OvCa cells with noggin/Fc alone decreased cell migration to below control (vehicle) levels. These data support the existence of an autocrine BMP signalling pathway that is capable of promoting OvCa cell motility.


Figure 4
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  		Fig. 4. BMP4 increases OvCa cell but not normal OSE cell motility. (A) OvCa cells were cultured onto etched grid cover slips, pre-treated with vehicle (upper panels) or BMP4 (lower panels) for 48 h, and a wounding assay was performed. The wound was monitored for a period of 12 h post-wound. Magnification x100. (B) The percentage of the total area covered by OvCa cells was assessed using image analysis software (Image J). The graph depicts data from a representative patient sample. Similar results were obtained using primary cells from 11 OvCa samples. Bars represent standard deviation from three independent experiments conducted in triplicate. *P < 0.05. (C) OvCa cells were cultured on etched grid cover slips, pre-treated with vehicle, BMP4, Ad-ALK3QD, BMP4 and noggin/Fc or noggin/Fc for 48 h, and a wounding assay was performed. Images depict the wound space at time 0 h (upper panels) and 12 h (lower panels). (D) Data were analysed as described in (B). Similar results were obtained using primary cells from three OvCa samples. (E) Normal OSE cells were pre-treated with vehicle, BMP4, BMP4 and noggin/Fc or noggin/Fc alone for 48 h, and a wounding assay was performed as described in (C). (F) The graph represents mean data from nine normal OSE samples. Bars represent standard deviation from one independent experiment on each of the nine samples conducted in triplicate.

 
To evaluate the motility response in normal OSE cells, wounding assays were conducted on nine different normal OSE cell samples. Despite the fact that normal OSEs respond to BMP4 by altering cell morphology (Figure 1A) and alter BMP4 target gene expression (10), we found no significant difference in motility between the control (vehicle) cells and any of the other treatments (Figure 4E and F). These results show that the motility of normal OSE cells was not affected by BMP4 or noggin/Fc in the same manner as OvCa cells, suggesting that OvCa cells have acquired the ability to enhance motility in response to BMP4.

BMP4 treatment or ALK3QD expression increases OvCa cell invasion
We also examined the effect of BMP4 ligand on OvCa cell invasion through a collagen I matrix. We found that in response to BMP4 invasion was increased when naive cells from three different patient samples were exposed to BMP4 during the invasion assay over an 18 h time period (Figure 5A and B). BMP4 pre-treatment (48 h) of these same primary OvCa cells produced different results; only one patient sample exhibited a significant increase in invasion, whereas no significant change was observed in two other patient samples. Further investigation would be required to determine why there is a differential effect on invasion in the two patient samples that do not respond to BMP4 pre-treatment, whereas the third patient sample maintained sensitivity. BMP receptor down-regulation is one possible explanation. These results are in contrast to a statistically significant increase in invasion when OvCa cells are transduced with an expression vector for a constitutively active BMP type I receptor (ALK3QD). Six different OvCa patient samples were tested and all showed significant increases in invasion over NT or Ad-GFP transduced cells (Figure 5C and D). No significant difference in invasion was seen between the NT and Ad-GFP-transduced OvCa samples. Furthermore, we conducted a modified Boyden chamber analysis using Transwell inserts (35) to determine whether BMP4 was chemotactic. Under no circumstances did BMP4 induce directional migration of primary OvCa cells (data not shown). These results support the notion that BMP4 signalling contributes to increased invasive capabilities of OvCa cells.


Figure 5
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  		Fig. 5. BMP4 treatment or ALK3QD expression increases OvCa cell invasion. OvCa cells were pre-treated with BMP4 for 48 h and at the time of the assay, or naive cells were treated with BMP4 at the time of assay and assessed for invasive capabilities through a collagen I matrix. (A) The average number of cells that invaded through the collagen I-coated filter is indicated for each treatment. The number of replicate assays for each sample is indicated. Data for three OvCa samples (76, 90 and 93) are shown. (B) Graphical representation of fold change in invasion relative to vehicle-treated cells that invaded, pooled from all OvCa samples tested, for each treatment. Bars represent standard deviation. *P < 0.05. (C and D) OvCa cells were (NT) or transduced with Ad-GFP and Ad-ALK3QD. (C) The average number of cells that invaded through the collagen I-coated filter is indicated for each treatment. The number of replicate assays for each sample is indicated. Data for six OvCa samples (64, 75, 85, 89, 92 and 101) are shown. (D) Graphical representation of fold change in invasion relative to Ad-GFP transduced cells that invaded, pooled from all OvCa samples tested, for each treatment. Bars represent standard deviation. *P < 0.05.

 
BMP4 treatment results in increased Rho GTPase activation
Alterations in the actin cytoskeleton, motility, adhesion and invasion typically involve activation of the Rho GTPases (36–38). To investigate whether BMP4 treatment induced Rho GTPase activation, Rac1, Rho and Cdc42 pull down assays were performed. These assays measure levels of activated, GTP-bound, Rho GTPases. In comparison with vehicle-treated cells, BMP4-treated cells show consistently higher levels of activated Rac1, Rho and Cdc42 between 1 and 4 h after treatment, and this activation decreased to baseline levels by 24 h (Figure 6). These results indicate that BMP4 signalling induced the activation of the small GTPases Rac1, Rho and Cdc42 in primary OvCa cells; however, the precise mechanism of Rho GTPase activation mediated by the BMP4 signalling pathway remains to be determined.


Figure 6
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  		Fig. 6. BMP4 treatment results in increased Rho GTPase activation. (A–C) Pooled data from four OvCa samples are shown. Cells were treated with BMP4 for up to 24 h (1, 2, 4 and 24 h post-BMP4 addition) and Rho GTPase activation (Rho, Rac1 or Cdc42) was assessed. Data are expressed as fold change in activation compared with time-matched vehicle-treated samples. Bars represent standard deviation from three independent experiments conducted in triplicate. *P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary data
 References
 
We determined that BMP4 signalling is able to affect cellular behaviours in primary human OvCa cells, including changes in morphology, adhesion, motility and invasion. BMP4 treatment of OvCa and normal OSE cells induces a significant increase in cell area in parallel with cytoskeletal re-organization. Interestingly, the BMP4-induced change in OvCa cell morphology is coincident with changes in markers associated with EMT. Furthermore, while BMP4 increases OvCa cell motility, normal OSE cell motility is not enhanced by treatment with exogenous BMP4. We also observed changes in the level of activated Rho GTPases (Rho, Rac1, Cdc42) in OvCa cells treated with BMP4. These data contribute to the notion that BMP signalling enhances OvCa cell aggressiveness.

BMP4 treatment of normal OSE and OvCa cells induces re-organization of the actin cytoskeleton into stress fibres throughout the cell, and in OvCa cells increases co-localization of F-actin with vinculin to membrane ruffles. Our data are consistent with BMP4 signalling causing a remodelling of the cellular machinery that is responsible for cellular morphology, adhesion, motility and invasion (36–38). BMP4 stimulation of OvCa cells changes the mRNA expression for various integrin receptors, ECM proteins and FAPs. Differential expression of adhesive proteins on OvCa cells in response to BMP4 likely mediates the increased adhesion and interaction with various ECM coatings. Blocking autocrine BMP4 signalling reduces OvCa cell adhesive properties, suggesting that endogenous BMP4 activity is important for cell–ECM interaction. OvCa metastasis is characterized by OvCa cell exfoliation from the primary tumour and diffusion throughout the peritoneal cavity, attaching and establishing micrometastases on peritoneal surfaces (39). Our results suggest that BMP4 affects the cells' ability to regulate adhesion proteins that allow them to adhere to diverse strata, which would be beneficial for OvCa metastatic progression.

BMP4-induced morphological changes point to the possibility that OvCa cells can be remodelled and are capable of transdifferentiating into a more mesenchymal phenotype. We observed that OvCa cells increase cellular motility and invasion in response to BMP4; these behaviours are associated with EMT (21,22). In correlation with these altered cellular behaviours, we found that mRNA and protein levels of Snail and Slug are elevated shortly after BMP4 treatment, whereas E-cadherin mRNA and protein levels decrease following 48 h of BMP4 treatment. Ectopic over-expression of Snail and Slug in the SkOV3 human OvCa cell line enhances their motility, invasiveness and tumorigenicity (40). In addition, we demonstrated that primary OvCa cells express smooth muscle actin and its immunolocalization was altered to resemble a mesenchymal pattern after BMP4 treatment. These features show that additional mesenchymal cell characteristics are acquired over a 48 h period in response to exogenous BMP4 treatment. Over-expression of ALK3QD results in similar morphological and behavioural changes as seen with exogenous BMP4 treatment. These results suggest that BMP4-induced effects are most probably produced by signalling through ALK3. Moreover, the rapid induction and increased magnitude of the cellular responses induced through ALK3QD is likely a reflection of the presence of a constitutively active receptor versus reliance on endogenous receptor dynamics that transmit signals with regulated amplitude. Similarly, exogenous BMP4 may produce EMT due to a higher level of signalling activity compared with autocrine BMP4 signalling, suggesting that OvCa cells require a certain threshold of signalling activity in order to induce EMT in vitro.

A putative function for autocrine BMP4 in OvCa cells was illustrated through blocking endogenous BMP4 signalling. Treatment of OvCa cells with noggin/Fc alone decreased both adhesion and motility in many cases to below control levels. These results are in agreement with the findings from Moll et al. (41) who demonstrated that transfecting established OvCa cell lines with an expression vector for the BMP inhibitor chordin, or using conditioned medium containing noggin, reduced cell motility and their ability to invade Matrigel (41). Together, these data support the argument that autocrine BMP4 may contribute to aggressive OvCa cell behaviour through enhancing motility and adhesion. Surprisingly, normal OSE motility is neither enhanced by treatment with exogenous BMP4 nor abrogated by noggin/Fc alone. This observation complements our previous finding showing that select BMP4 target gene expression, e.g. ID1 and ID3 proto-oncogenes, is more pronounced in OvCa cells compared with OSE cells (10). The molecular basis of these differential responses remains unknown; however, we have determined that the mRNA for BMPRII and ALK3 and Smad1 and Smad5 protein levels remain unchanged between OSE and OvCa cells (B.L.Thériault, T.G.Shepherd and M.W.Nachtigal, unpublished observations). Collectively, these data suggest that a component of the BMP4 signalling pathway, an interacting signalling pathway, or a downstream effector molecule is altered in OvCa cells. These distinct cellular responses begin to provide insight into how autocrine BMP4 may contribute to OvCa etiology.

BMP signalling is classically mediated via a Smad cascade that is activated by the BMP receptor complex. Recent studies have demonstrated that BMP signalling can also be mediated through alternative intracellular mediators, including Cdc42 and LIM Kinase 1 (LIMK1) (42,43). Rho GTPases act upstream of LIMKs to regulate actin polymerization. While the pathway leading from the activated BMP receptor complex to Cdc42 remains undetermined, these investigators have identified a direct interaction of LIMK1 with the C-terminal tail of BMPRII. LIMK1 is activated by Cdc42 following BMP activation of the BMP receptor complex. Activated LIMK1 remains associated with the activated BMP receptor complex, and is capable of phosphorylating its downstream effector cofilin. Cofilin is a member of the F-actin-binding protein family that regulates actin dynamics. We show that Cdc42, along with Rac1 and Rho, is activated shortly after BMP4 treatment of OvCa cells, which precedes the observed morphological changes associated with actin remodelling. Interestingly, elevated Rho expression has been correlated with more aggressive OvCa behaviour (44,45). In agreement with Lee-Hoeflich et al. (42), our results point to the role of BMP signalling in activating modulators of actin cytoskeletal dynamics, the Rho GTPases. In light of these results, we investigated whether LIMK activity in OvCa cells was altered in response to BMP4 stimulation. In experiments using cell lysates from two patient samples, we measured LIMK1 activity in BMP4-treated OvCa cells from 30 min to 24 h and determined that there was no alteration in cofilin phosphorylation (data not shown). Moreover, the phenotypic responses of primary OvCa cells to BMP4 treatment can be replicated using an activated type I receptor, ALK3QD, which signals independent of the BMPRII. To our knowledge, there is no evidence that LIMK1 interacts with BMP type I receptors. Therefore, these data suggest that LIMK1 is unlikely to participate in the BMP4-induced cellular responses in OvCa cells.

We observe that Rho GTPase activation peaks between 1 and 4 h after a single treatment with BMP4 and returns to baseline levels of activity by 24 h; however, BMP4-induced alterations in cell morphology, adhesion, motility and invasion are observed after ~48 h. Additional experiments were conducted using three different patient samples for up to 72 h post-BMP4 treatment. In all cases, Rho GTPase activation had returned to baseline by 24 h and remained at baseline levels up to 72 h after a single treatment with BMP4 (data not shown). We hypothesize that the transient increase in Rho GTPase activity is one of the initiating events leading to cellular remodelling resulting in cells possessing these altered traits. Furthermore, enhanced migration was only observed following BMP4-induced morphological alterations. Indeed, no increases in cell motility above control levels was observed when naive OvCa cells were treated with BMP4, or when OvCa cells were pre-treated with BMP4 for 24 h prior to an observed change in cellular morphology (data not shown). These data show that despite small Rho GTPase activation in response to BMP4 initially, this event alone is insufficient to augment motility rates in naive OvCa cells. Therefore, morphological alterations appear to be a prerequisite for enhanced motility, which is consistent with BMP4 inducing an EMT in OvCa cells (46).

Rho GTPases are well known to participate in cellular processes of adhesion, motility and invasion (46,47). While we believe that Rho GTPases probably play a part in the cellular responses that we have presented, our experimental conditions preclude measuring Rho GTPase activity during the assessment of cellular motility and adhesion. This is due to the fact that small Rho GTPase activation assays are conducted under low-serum conditions (0.2% FBS) in order to increase the sensitivity of the assay. In our experience, assessment of Rho GTPase activation when cells are maintained in high serum (10% FBS) produces a high background level of activation. Under normal growth conditions, BMP4 pre-treatment promotes enhanced adhesion and migration, but given that these experiments are conducted in the presence of 10% FBS, we cannot directly determine if Rho GTPase activation is also increased under these experimental conditions. However, use of the Rho kinase inhibitor Y-27632 and the Rac1 inhibitor NSC23766 in the presence or absence of BMP4 under normal growth conditions resulted in complete abrogation of cellular motility (data not shown), strongly suggesting that the Rho GTPases are involved in this activity. It remains to be determined how BMP4 is causing the changes in OvCa cell behaviour, whether these responses are dependent upon Smad signalling and how other mediators such as the Rho GTPases are connected to the BMP4 signalling cascade.

BMP4 signalling affects a wide variety of cellular behaviours in OvCa cells, leading us to postulate that BMP4 signalling has the ability to promote OvCa cell aggressiveness, which may translate into increased metastatic potential and poor patient outcomes. Overall, our studies suggest a link between autocrine BMP signalling mediated through the Rho GTPase family and Snail- and Slug-induced EMT that collectively contributes to enhanced OvCa cell adhesion, motility and invasiveness. The fact that BMP4-induced motility and adhesion can be blocked with a BMP signalling inhibitor (noggin) provides a rationale to develop and test anti-BMP therapeutics (48,49) to prevent or stop OvCa progression.


   Supplementary data
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary table SI and figures S1, S2 and S3 can be found at http://carcin.oxfordjournals.org/


   Acknowledgments
 
We would like to thank Dr J. Blay (Dalhousie University) for the critical appraisal of this manuscript. We would also like to thank Drs L. Attisano (University of Toronto) and B.C. Vanderhyden (Ottawa Health Research Institute) for various DNA plasmids used in these studies, and Dr P. Marignani (Dalhousie University) for helpful discussions. This work was supported by a grant to M.W.N. from the Canadian Cancer Society (National Cancer Institute of Canada Grant no. 15303). B.L.T is a Nova Scotia Health Research Foundation Student, T.G.S. was a Research Fellow of the Terry Fox Foundation awarded from the National Cancer Institute of Canada and M.L.M is the Rossetti Fellow for Cancer Research with an award administered through the Dalhousie Medical Research Foundation and the Cancer Research Training Program. M.W.N. is a Research Scientist of the Canadian Cancer Society awarded from the National Cancer Institute of Canada.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 References
 

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Received October 2, 2006; revised January 12, 2007; accepted January 15, 2007.


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