Constitutive activation of BMP signalling abrogates experimental metastasis of OVCA429 cells via reduced cell adhesion
© Shepherd et al. 2010
Received: 7 November 2009
Accepted: 26 February 2010
Published: 26 February 2010
Activation of bone morphogenetic protein (BMP)4 signalling in human ovarian cancer cells induces a number of phenotypic changes in vitro, including altered cell morphology, adhesion, motility and invasion, relative to normal human ovarian surface epithelial cells. From these in vitro analyses, we had hypothesized that active BMP signalling promotes the metastatic potential of ovarian cancer.
To test this directly, we engineered OVCA429 human ovarian cancer cells possessing doxycycline-inducible expression of a constitutively-active mutant BMP receptor, ALK3QD, and administered these cells to immunocompromised mice. Further characterization was performed in vitro to address the role of activated BMP signalling on the EOC phenotype, with particular emphasis on epithelial-mesenchymal transition (EMT) and cell adhesion.
Unexpectedly, doxycycline-induced ALK3QD expression in OVCA429 cells reduced tumour implantation on peritoneal surfaces and ascites formation when xenografted into immunocompromised mice by intraperitoneal injection. To determine the potential mechanisms controlling this in vivo observation, we followed with several cell culture experiments. Doxycycline-induced ALK3QD expression enhanced the refractile, spindle-shaped morphology of cultured OVCA429 cells eliciting an EMT-like response. Using in vitro wound healing assays, we observed that ALK3QD-expressing cells migrated with long, cytoplasmic projections extending into the wound space. The phenotypic alterations of ALK3QD-expressing cells correlated with changes in specific gene expression patterns of EMT, including increased Snail and Slug and reduced E-cadherin mRNA expression. In addition, ALK3QD signalling reduced β1- and β3-integrin expression, critical molecules involved in ovarian cancer cell adhesion. The combination of reduced E-cadherin and β-integrin expression correlates directly with the reduced EOC cell cohesion in spheroids and reduced cell adhesion to the extracellular matrix substrates fibronectin and vitronectin that was observed.
We propose that the key steps of ovarian cancer metastasis, specifically cell cohesion of multicellular aggregates in ascites and cell adhesion for reattachment to secondary sites, may be inhibited by overactive BMP signalling, thereby decreasing the ultimate malignant potential of ovarian cancer in this model system.
Ovarian cancer is the most lethal of the gynaecologic malignancies in the Western world. The majority of ovarian cancers are detected as late-stage disease and involve the dissemination of tumour cells throughout the peritoneal cavity and the production of ascites; these clinical assessments are correlated with a very poor prognosis (only 5-40% five-year survival) for patients. Successful early detection and more effective management of late-stage disease are crucial to improving the survival and quality-of-life of ovarian cancer patients. Understanding the underlying molecular mechanisms of ovarian cancer pathogenesis is key to achieving this goal.
Previous work from our laboratory demonstrated that normal human ovarian surface epithelial (OSE) cells and human epithelial ovarian cancer (EOC) cells possess an intact autocrine bone morphogenetic protein-4 (BMP4) signalling pathway. BMPs comprise approximately 20 unique members of the transforming growth factor-beta (TGFβ) superfamily of cytokines. BMPs act as extracellular dimeric ligands by binding to the type I (ALK2, ALK3, and ALK6) and type II (BMPR2) receptors. BMP signalling is mediated via a heterotetrameric receptor complex composed of a type I receptor that is phosphorylated at the intracellular GS domain by type II receptor serine/threonine kinase activity, leading to the association of receptor-activated Smad (R-Smad) proteins. Upon phosphorylation, the BMP-specific R-Smads (Smads 1, 5 and 8) dimerize and associate with the common-mediator Smad, Smad4. This activated Smad complex translocates to the nucleus and regulates the transcription of target genes, typically via its interaction with several other transcription factors and co-activator and co-repressor proteins. Recent work also shows that Smad independent signalling can be initiated by the activated receptor complex[4, 6, 7].
The multifaceted and complicated roles of TGFβ signalling in the pathogenesis of many human cancers is well established[8, 9], yet our understanding of the contribution of BMP signalling to cancer biology is limited. In many instances, activation of BMP signalling inhibits cell growth and induces apoptosis in different cancer cell types[10–16]. The identification of inactivating germline mutations in the human BMPR1A gene (encoding the ALK3 receptor) in juvenile polyposis patients indicates a putative tumour suppressor function of active BMP signalling in colon cancer[17, 18]. However, other studies have found that BMP signalling may be implicated in increasing metastatic potential[19–21] and tumour angiogenesis. While there has been some advancement in our understanding of the functional implications of BMP signalling in various cancers, the contribution of BMP signalling to ovarian cancer pathogenesis requires further clarification.
Treatment of primary human OSE and EOC cells with exogenous BMP4 ligand results in a cell spreading phenotype and increased cell adhesion[2, 23]. Furthermore, BMP4 induces an epithelial-mesenchymal transition (EMT) morphologic response in primary EOC cells isolated from patient ascites by increasing Snail and Slug expression and a subsequent decrease in E-cadherin . BMP4 directly upregulates ID1 and ID3 proto-oncogene expression in EOC cells compared with normal OSE, and BMP4 signalling increases EOC cell motility whereas normal OSE cell motility is unaffected. We sought to develop a model system to elucidate the function of active BMP signalling in ovarian cancer metastasis. We chose to use the human ovarian cancer cell line OVCA429 which is capable of producing ascites and peritoneal implants mimicking the spread of ovarian cancer observed in patients . To this end, we employed a doxycycline (Dox)-inducible expression system in OVCA429 cells to ectopically express a constitutively-active mutant BMP type I receptor (ALK3QD). Herein, we report that constitutively-active ALK3 receptor signalling decreases the intraperitoneal dissemination of OVCA429 cells in nude mouse xenografts. We provide further evidence that this may occur via downregulation of E-cadherin and β1-/β3-integrin expression thereby reducing cell-cell cohesion and cell-substratum adhesion. The application of this model system to an in vivo context provides insight into how cellular responses affected by constitutive BMP signalling directly impacts ovarian cancer metastasis.
CaOV3 and SkOV3 human ovarian cancer cells were grown in Dulbecco's modified Eagle medium (Invitrogen) containing 10% heat-inactivated fetal bovine serum (FBS; Hyclone). OVCA429 human ovarian cancer cells (gift of Dr. B. Vanderhyden, Ottawa Regional Cancer Centre) were grown in Minimal essential medium Eagle-alpha (Invitrogen) containing 10% FBS and supplemented with 0.1 mM non-essential amino acids (Invitrogen). Primary cultures of human OSE and EOC cells were isolated and maintained as previously described. Treatment of OVCA429 cells with recombinant human BMP4 (R&D Systems) was performed as described previously. 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).
OVCA429 Tet-On cells (429T cells) were generated by transfecting OVCA429 cells with pTet-On vector (Clontech) using GeneJuice reagent (Novagen), followed by selection with 1 mg/mL Geneticin™ (Invitrogen). Of the 27 separate clones generated, 5 clones displayed detectable doxycycline-inducible activity, as assessed by transient transfection of the pTRE2-luc vector and followed by luciferase assays performed as previously described. Doxycycline hyclate (Dox; Sigma) was used at a concentration of 2 μg/mL in all experiments, unless otherwise indicated. Dox-inducible ALK3QD-expressing cells (429T-ALK3QD) were generated by transfecting 429T cells with pTRE2-ALK3QD-HA (original pCMV5-Alk3QDHA plasmid was a gift from Dr. L. Attisano, U. of Toronto), and selection with 250 μg/mL Hygromycin B (Invitrogen). Five different 429T-ALK3QD cell lines possessing Dox-inducible ALK3QD expression were identified by Western blot analysis, and two independent clones (clones 429T-A44 and -54) were chosen for further examination. Subsequent growth of 429T and 429T-ALK3QD cell lines was maintained using 100 μg/mL Geneticin™ and 100 μg/mL Hygromycin B.
Northern analysis was performed as previously described, using radiolabelled probes synthesized from human cDNA fragments of ALK3 (nucleotides 145-594), ALK6 (nucleotides 907-1256), BMPR2 (nucleotides 2561-3010), GAPDH (nucleotides 270-620), ID1 (nucleotides 189-549), and ID3 (nucleotides 356-750) as templates.
Protein isolation and subsequent Western analyses were performed as previously described. ALK3QD protein expression was detected using anti-HA-Peroxidase 3F10 monoclonal antibody (1:1000 dilution; Roche). The anti-actin polyclonal antibody (1:1000 dilution; Sigma) followed by incubation with horseradish peroxidase-conjugated sheep anti-rabbit IgG secondary antibody (1:5000 dilution; Chemicon) was used as a control for protein loading.
Tumour cell xenografting
All studies conformed to the approved animal utilization protocol and Canadian Council for Animal Care guidelines. Eight- to 10-week-old female CD-1 nu/nu athymic nude mice (Charles River) were maintained in a sterile barrier animal facility. After approximately one week from receipt, 30 mice were started on a diet of gamma-irradiated rodent chow containing Dox at a concentration of 1000 ppm (Research Diets) for 2 d, and another group of 30 mice remained on the in-house rodent chow diet. Mice were supplied with food and water ad libitum. Each mouse was injected i.p. with a suspension of 5 × 105 cells (either 429T-ALK3QD or 429T control) in a volume of 0.2 mL sterile 1 × PBS, resulting in four groups of fifteen mice (429T-ALK3QD or 429T controls, with or without Dox treatment). Mice were monitored for 90 d post-injection for signs of ascites formation or visible morbidity. At the experimental endpoint (90 d), mice were sacrificed and dissection was performed to assess and quantify ascites production and tumour formation.
Tissues harvested for histological analysis were fixed immediately in 4% paraformaldehyde/1 × PBS, paraffin-embedded and sectioned at 5 μm then stained with haematoxylin and eosin (tissue processing performed by Molecular Pathology, Robarts Research Institute, UWO). Microscopic images of stained tissue sections were captured using an Olympus IX70 inverted microscope and Image Pro 6.2 software, and subsequently adjusted for brightness/contrast and colour balance using Adobe Photoshop 7.0 software.
Confirmation of ALK3 QD transgene expression in tumours that formed in nude mice was performed by RT-PCR analysis of total RNA isolated from homogenized tissue using the Total RNA isolation kit (Sigma) as per manufacturer's instructions. Reverse transcription was performed with 2 μg of RNA reverse transcribed into cDNA using oligo-dT decamers as primers and Superscript II reverse transcriptase (Invitrogen) as per manufacturer's instructions. Subsequent PCR was carried out using oligonucleotides that specifically amplify the 3' end of the ALK3QD-HA cDNA construct. Human GAPDH mRNA expression served as a control for tumour xenograft material in each sample.
For quantitative RT-PCR analysis, total RNA was isolated from cells treated with 2 μg/mL Dox for 2 days, or left untreated, and 2 μg of RNA was subsequently reverse transcribed into cDNA using oligo-dT decamers as primers and Superscript II reverse transcriptase (Invitrogen) as per manufacturer's instructions. PCR was performed using the Brilliant SYBR green QPCR Master Mix (Stratagene), and real-time measurement of the PCR reactions was recorded using the Mx3000P Real-time PCR System (Stratagene), and quantified using the 2-ΔΔCt method; GAPDH expression was used for normalization, and the fold change in mRNA expression was calculated versus untreated cell samples.
All human gene-specific primer sequences used in RT-PCR are available upon request.
Scratch wound assays
Cells were seeded at 2 × 105 cells in 6-well dishes and 24 h later were treated with 2 μg/mL Dox, or left untreated. Cells were grown for 2-3 d until confluent monolayers were achieved, then a wound was created by scratching the wells with a sterile plastic pipette tip (~1 mm space). After several gentle washes with 1 × PBS, media was replaced (with or without Dox) and cells were monitored at multiple timepoints and photographed at 24 h. Photographic images were captured using a Nikon Coolpix digital camera and Nikon inverted phase contrast microscope at 100 × magnification.
For assessment of cell detachment, cells were seeded at 5 × 105 cells in 6-well dishes, and 24 h later were treated with 2 μg/mL Dox, or left untreated. Detached cells were quantified as previously described.
In adhesion experiments, cell lines were treated with 2 μg/mL Dox, or left untreated, and grown for 2-3 d until confluent monolayers were achieved. Adhesion assays were performed as previously described. Cells were plated at a density of 1 × 105 cells/well into 24-well dishes previously coated with 500 ng/cm2 fibronectin or 200 ng/cm2 vitronectin (Sigma), and blocked with 1% BSA.
429T and 429T-ALK3QD cells were pre-treated with 2 μg/mL Dox for 2 days, or left untreated, while grown in monolayer culture. One hundred thousand cells/well were then seeded in quadruplicate into a 24-well Ultra-Low Attachment cluster plate (Corning) and the same culture conditions (i.e. +/- Dox) were maintained during spheroid formation. Images were captured at the 2-day timepoint using an Olympus IX70 inverted microscope at 100 × magnification and Image Pro 6.2 software.
Statistical analyses were performed using the nonparametric Mann-Whitney test with 95% confidence intervals (GraphPad Prism 4). Values of significance are indicated in the Legends to Figures.
BMP receptor expression in OSE and EOC cells
Constitutive ALK3 receptor signalling reduces metastatic potential in vivo
Summary of ovarian cancer cell xenograft studies in nude mice.
No. of mice with ascites (%)
Mean ascites volume (± SEM)
1.5 (± 0.9)
1.0 (± 0.6)
0.7 (± 0.4)
1.1 (± 0.5)
No. of mice with tumours (%)
Mean tumour number (± SEM)
7.5 (± 2.7)
10.4 (± 3.6)
6.2 (± 2.0)
2.2 (± 1.3)
Tumour sites (number of mice)b
Constitutive ALK3 receptor signalling reduces EOC cell adhesion in vitro
Cell adhesion is likely a key checkpoint regulating the efficient spread of ovarian cancer cells during metastasis. Potential molecular targets of ALK3QD signalling mediating the observed reduction in ovarian cancer cell adhesion are genes encoding integrins and/or extracellular matrix (ECM) components. We performed gene expression analyses for several specific integrins and ECM components known to be expressed by OSE and EOC cells[31, 32]. Dox-treated 429T-ALK3QD cells possessed a 64% decrease in ITGB1 (β1-integrin) and 43% decrease in ITGB3 (β3-integrin) mRNA expression versus 429T controls (Fig. 4). In addition, we observed a reciprocal elevated expression of ECM genes FN1 (fibronectin) and VTN (vitronectin) mRNA in ALK3QD-expressing cells by 49% and 69%, respectively.
To address whether this altered phenotype in response to ALK3QD expression affected the motility of the cells, standard in vitro wound-healing assays were performed. There was no difference in motility rates among Dox-treated and untreated 429T, 429T-A44 and 429T-A54 cells (data not shown). When cells were observed during the progress of cell migration (i.e. at 24 hours after wounding), Dox-treated 429T-A44 and 429T-A54 cells possessed a more spindle-shaped morphology as they migrated into the manufactured wound space with cells possessing long, cytoplasmic projections extending in the direction of cell movement (Fig. 5B). Interestingly, 429T and 429T-ALK3QD cells are poorly invasive through matrices composed of either rat tail collagen or gelatin, regardless of Dox-treatment (data not shown). This lack of invasiveness is identical to the parental OVCA429 cell line (Mujoomdar & Nachtigal, unpublished observations). Taken together, these data provide further validation that ALK3QD expression in OVCA429 cells results in morphological changes paralleling the observed alterations in EMT marker expression.
Primary cultures of normal human OSE cells and EOC cells possess an intact BMP4 signalling pathway, yet there are important differences between the response of normal OSE cells to exogenous BMP4 and that of EOC cells [2, 23]. For example, primary human EOC cells achieve higher levels of BMP4-induced ID1 and ID3 proto-oncogene expression than do normal human OSE cells (~10-15 fold in EOC cells, compared to 2-3 fold in normal OSE). The differential response to BMP4 signalling between OSE and EOC cells is unlikely to be due to altered BMP4 receptor expression levels since we observed no significant differences in the mRNA level of ALK3 or BMPR2, and expression of ALK6 mRNA was largely undetectable in all primary cell samples. Additionally, BMPR2, Smad1 and Smad5 protein levels were similar in primary OSE and EOC cell samples (Shepherd & Nachtigal, unpublished observations).
To observe the effect of autonomous BMP signalling in EOC cells, we chose to express the constitutively-active mutant BMP type I receptor ALK3QD in OVCA429 ovarian cancer cells. Several studies have used mutant BMP receptor expression to obtain insight into the role of BMP signalling in human cancer cells. For example, dominant-negative BMPR2 affects the growth of human breast cancer cells in vitro by blocking cells in G1 of the cell cycle. Constitutively-active ALK6QD receptor expression decreases the proliferation of human prostate cancer cells as well as their ability to form tumours in nude mice, whereas blocking BMP signalling by expression of dominant-negative BMPR2 enhances prostate cancer tumorigenicity. No mutant BMP receptors have been identified in primary human EOC cells, however an intact autocrine BMP4 pathway exists to induce EOC cell spreading, and increased adhesion, motility and invasion[2, 23]. Moreover, cell migration is greatly reduced by blocking this autocrine BMP4 signalling pathway with the BMP2/4 antagonist Noggin . Established human EOC cell lines are responsive to exogenous BMP4 ligand at the level of target gene expression, yet appear particularly unaffected in terms of other phenotypic changes observed in primary EOC cell samples from patients[2, 23]. By utilizing the inducible expression of a constitutively-active ALK3QD receptor in OVCA429 ovarian cancer cells, we provide additional evidence for the contribution of BMP signalling to affect cellular morphology and adherence in EOC cells, and now extend these findings with direct analysis of its impact on EOC metastasis in vivo.
In this report we demonstrate that constitutive BMP signalling through the mutant ALK3 receptor induces EMT markers, consistent with our findings observed in primary EOC cells. EMT is commonly associated with aggressive cancer cell behaviour. Indeed, ectopic overexpression of Snail and Slug in the SkOV3 human ovarian cancer cell line enhances their motility, invasiveness and tumorigenicity. The precise role for EMT in ovarian cancer pathogenesis, however, is not straightforward . Although decreased E-cadherin expression is a hallmark of EMT and is usually correlated with a higher degree of malignancy in most other carcinomas, forced overexpression of E-cadherin in immortalized human OSE cells enhances pre-neoplastic features in vitro and establishes tumour-forming ability in vivo [41, 42]. Our data independently supports this finding since ALK3QD expression led to morphological alterations and changes in gene expression consistent with EMT in vitro, yet resulted in a decreased ability to produce ascites and form tumours in vivo. Perhaps the functional role of EMT in ovarian cancer is an exception to norm among epithelial-derived malignancies and represents a key process underlying the unique mode of metastasis, i.e. direct dissemination into the peritoneal cavity, observed in this type of carcinoma[1, 33].
ALK3QD-expressing OVCA429 cells exhibited decreased ability to form spheroids as well as reduced cell-substratum adhesion with concomitant alterations in the expression of a number of genes encoding integrins and ECM components. Le Page and colleagues have recently demonstrated that BMP2 treatment of several ovarian cancer cell lines also reduces the cell-cell cohesion during spheroid formation ; however, evidence for the molecular mechanism was not presented. BMP signalling has been shown to alter the expression of integrins and their substrates present in the ECM in several cell types, including EOC cells. For example, α5β1 and αvβ3 expression is increased in osteoblasts in response to BMP signalling resulting in increased mesenchymal cell adherence[44–46]. Altered expression of β-integrins has numerous implications in human cancer pathogenesis. Conditional loss of β1-integrin reduces mammary tumour formation in transgenic mice. Specific to ovarian cancer, it has been postulated that β1-integrin expression by EOC cells functions during metastasis to promote cell adhesion to the peritoneal mesothelial surfaces during tumour implantation[48–50]. In addition, blocking β1-integrin with interfering antibodies disaggregates EOC spheroids and reduces cell adhesion, spheroid formation, and attachment to peritoneal surfaces [48, 51]. Coordinated expression of β3-integrin is also involved in EOC cell adhesion, and β3-integrin interaction with vitronectin via αv-integrin promotes EOC cell proliferation, motility, and ECM degradation[52, 53]. In contrast, Kaur and colleagues recently demonstrated that forced αvβ3-integrin overexpression in SkOV3ip1 cells increases cell adhesion in vitro yet reduces both invasion and their ability to form secondary tumours in mice; clinical data from this same report implicates that αv/β3-integrin expression may represent a favourable prognostic marker in ovarian cancer . Ascites-derived primary human EOC cells treated with BMP4 leads to increased β1- and β3-integrin mRNA expression and correlates with increased adherence to a variety of ECM substrates in vitro . In this report, we propose that the downregulation of β1- and β3-integrins caused by an aberrant constitutively-active BMP signalling pathway in a more malignant variant of EOC cells (i.e. the OVCA429 cell line) decreases cell adhesion in vitro and thereby leads to reduced ascites and intraperitoneal tumour formation in vivo. Whether BMP signalling regulates β1- and β3-integrin expression directly or indirectly to affect EOC cell adhesion during specific steps of ovarian tumorigenesis requires further investigation. The recent work by Kaur et al specifically examined ectopic overexpression of β3-integrin in SkOV3ip1 cells, whereas other studies have evaluated endogenous integrin expression and function within several other EOC cell lines (CaOV3, SkOV3, OVCAR3, OVCAR5, SW626, OV-MZ-6, 36M2)[48–53]. The majority of these studies suggest that intact integrin function inherent to EOC cells is necessary for adhesion to ECM and peritoneal surfaces in the promotion of EOC metastasis. From our standpoint, it is imperative to perform future studies using both ascites-derived primary EOC cells and established EOC cell lines to help clarify the mechanisms underlying the observed differences in integrin-mediated cell adhesion on the malignant behaviour among these cell types.
Differential effects of BMP signalling in vitro compared to in vivo have been observed in other tumour models. For example, Langenfeld and colleagues demonstrate that BMP2 induces human lung adenocarcinoma A549 cell proliferation in vitro in the presence of serum; but when injected into nude mice BMP2-expressing A549 cells have reduced subcutaneous tumour growth, while development of lung metastases is augmented. They suggest that the cellular response to BMP signalling is dependent upon additional factors in specific tumour microenvironments. Our model has ALK3 signalling constitutively maintained in a cell-autonomous fashion thereby impacting OVCA429 cells directly. From this, we propose that the decreased EOC cell adhesion observed in vitro is a critical factor contributing to reduced intraperitoneal ascites and tumour formation in vivo as compared with control cells. It will be imperative to further investigate the functional impact of BMP ligands and antagonists on EOC cells versus the surrounding tissue microenvironment during ovarian tumour formation and metastasis.
In summary, we propose that dysregulated BMP signalling is implicated in EOC cell exfoliation from the primary tumour site and subsequent dissemination during ovarian cancer progression. Overactive BMP signalling mediates efficient ovarian cancer cell detachment from the primary tumour; however, at later steps of ovarian cancer progression, down-regulation of BMP signalling may be necessary to establish tumour cell adherence and secondary metastasis formation. To this end, we are currently developing additional in vivo models of ovarian cancer to address the implication of modulated BMP signalling on each step from initiation to metastasis.
We would like to thank Drs. Brigitte Thériault and Gabriel DiMattia for critical appraisal of this manuscript. We would also like to thank the Dr. Liliana Attisano for the ALK3QD cDNA plasmid, and Dr. Barbara Vanderhyden for the OVCA429 cell line. This work was supported by grants to M.W.N. from the Canadian Cancer Society (National Cancer Institute of Canada Grant # 15303) and T.G.S from The Cancer Research Society. T.G.S. was a Research Fellow of the Terry Fox Foundation awarded from the NCIC. M.W.N. was a Research Scientist of the Canadian Cancer Society awarded from the NCIC.
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