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Although a dichotomous role for TGF-β in carcinogenesis is well-established, this is not the case for BMP signalling. More in vitro and in vivo evidence is required to determine whether this pathway is indeed oncogenic or tumour suppressive and in which

types and stages of cancers this is true. BMP signalling is generally considered a critical pathway during development controlling cell differentiation. Therefore, it is reasonable to suppose that induction of BMP signalling in cancer cells might be anti-oncogenic causing a differentiation-related program to force cancer cells out of the cell cycle. It is also possible that BMP signalling has been co-opted by cancer cells in a pro-oncogenic manner to facilitate maintenance of the cancer cell phenotype.

Our study focused on dissecting the contribution of the BMP signalling cascade during the various phases of ovarian cancer progression using a biologically-relevant, tractable in vitro model system. This model takes into account the unique mode of ovarian cancer metastasis, whereby multicellular spheroids represent an important conduit through which cells are able to survive until they reach a mesothelial surface where re-implantation and invasion are possible. It has been suggested that ovarian cancer cells within multicellular aggregates or spheroids undergo EMT, acquiring more mesenchymal characteristics, preparing them to reattach and invade, forming secondary metastases when conditions are favourable4,5.

The data presented in Chapter 2 confirms that spheroids formed from primary ovarian cancer cells undergo EMT, characterized by decreased expression of the E- cadherin gene. This is supported by previous evidence that E-cadherin expression is reduced in cells isolated from ascites compared to their solid tumour counterparts6 as well as other studies demonstrating that ovarian cancer cell lines with more mesenchymal characteristics have an increased propensity for compact spheroid formation5_{. The} autonomous down-regulation of BMP signalling during spheroid formation and the decreased propensity for compact spheroid formation when this pathway is activated suggests that BMP signalling may in fact be opposing the natural EMT response of EOC spheroids. This is concordance with a study published in 2011 by the Weinburg lab in which they documented that the BMP pathway had the ability to antagonize TGF-β-

induced EMT in mammary epithelial cells7_{. Indeed, activation of BMP signalling within}

EOC spheroids results in increased E-cadherin expression when compared to controls, suggesting that inhibition of EMT could be a potential mechanism through which BMP signalling decreases spheroid compaction.

Although activated BMP signalling in spheroids results in smaller, more loosely aggregated clusters this does not result in an overall decrease in cell viability. Therefore, it should not be assumed that activation of this pathway is detrimental. Rather, it may ‘prime’ cells within these aggregates to more readily attach and disperse. Indeed, when spheroids with constitutively active BMP signalling are exposed to an adherent substratum, they have an increased propensity for reattachment and dispersion. This is supported by previous work demonstrating that BMP4 has the ability to enhance motility and invasion of adherent primary ovarian cancer cells8,9.

Taken together, these findings suggest that inhibition of BMP signalling may in fact be a viable therapeutic target through which to prevent EOC spheroid reattachment and formation of secondary metastatic lesions. We show that inhibition of the BMP pathway using a small molecule inhibitor as well as BMP antagonist, noggin, does in fact decrease the ability of cells to disperse from a spheroid following re-introduction to an adherent substratum. Since this does not completely prevent spheroids from reattaching, it is likely that the BMP signalling pathway acts in conjunction with other pathways during this process.

Our studies uncovered an interaction between the BMP and AKT signalling cascades and demonstrated that these two pathways act in concert to promote EOC spheroid reattachment. This finding is supported by other reports demonstrating that the PI3K/AKT pathway is required for BMP-induced migration and invasion in gastric, colon and pancreatic cancer cells10,11_{. This highlights the potential for targeting these pathways} in combination for treatment of metastatic ovarian cancer. Xenograft models will be important to determine whether this interaction is translatable in an in vivo setting. Weroha and colleagues recently published elegant work validating the use of a novel ‘tumourgraft’ model whereby ovarian cancer patient tumour material is minced and

intraperitoneally injected into immune-compromised mice12. The tumours formed in mice

recapitulate the clinical and molecular characteristics of the patient tumour but also mimic the patient’s clinical response to chemotherapeutic treatment. This is an extremely exciting, highly translatable in vivo model with which to test the potential response of an patient’s tumour to a targeted therapeutic. Using this model, we would be able to test the

therapeutic response of a number of ovarian cancer tumour specimens to treatment with the small molecule inhibitors of BMP and AKT signalling that I have in the studies described in this thesis. Molecular analysis of these ‘tumourgrafts’ would allow us to determine patient-specific responses to modulation of these two signalling pathways prior to and following treatment.

This work highlights the fact that the BMP signalling pathway exerts the majority of its effects during spheroid reattachment and dispersion. Therefore, it would be interest to examine the contribution of tumour-stroma interactions in this process and the role that the BMP signalling pathway plays in mediating these interactions. Carcinoma-associated fibroblasts (CAFs) have become increasingly recognized as important components of the tumour microenvironment that can aid in the initiation and progression of a number of different cancers including breast, prostate and ovarian13-16. Interestingly, normal fibroblasts co-cultured with carcinoma cells results in an irreversible conversion to a CAF phenotype, suggesting that fibroblasts exposed to cancer cells exhibit permanent, heritable changes13,14,16. Recent studies in ovarian cancer have demonstrated that the presence of CAFs can contribute to tumour progression, omental and lymph node metastases, in addition to being associated with poor patient prognosis17,18. Fu and colleagues were able to obtain primary cultures of CAFs from ovarian cancer patients and

co-culture these with EOC cells19. Analysis of conditioned medium from this co-culture

system revealed the presence of number of soluble factors including TGF-β and a number of BMPs. Using our in vitro system, it would be of interest to determine the effects of this co-culture media on spheroid reattachment both under ambient conditions as well in spheroids with constitutively activated BMP signalling and those treated with noggin. These studies would provide an additional layer of complexity to our model system, allowing us to not only determine the contribution of CAFs to spheroid reattachment and dispersion but also allow us to determine the interaction between the BMP signalling pathway and this population of cells (Figure 4.1).

The results presented in this thesis have uncovered an important role for BMP signalling in cellular cohesion and EMT during spheroid formation and cellular motility

during reattachment. This has laid the groundwork for preclinical models to investigate the utility of targeting this pathway for treatment of advanced-stage ovarian cancer.

4.3

LKB1 has AMPK-independent effects on cellular