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BMPR2, Smad1 and Smad5 protein levels were similar in primary OSE and EOC cell samples (Shepherd & Nachtigal, unpublished observations).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.