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High grade serous ovarian cancer (HGSOC) is the leading cause of death among all gynaecological malignancies. Despite recent advances in maximal effort surgery and systematic treatment, the 5-year survival remains as low as 35-40%. The response to treatment is heterogeneous even among the HGSOC population, although most of the patients are treated without stratification. It is therefore crucial to identify novel predictive and prognostic biomarkers to guide appropriate treatment, and discover novel therapeutic options for this lethal disease. Medical images, including computed tomography (CT) scans, are often used as a diagnostic tool during HGSOC treatment. In the first part of this project, we hypothesised that prognosis- relevant information could be hidden within CT scans and may be used as novel biomarker in HGSOC. We thus extracted 657 image features from each CT scan using a customised software; we then successfully generated and validated a prognostic model, based on a weighted four-feature signature. This CT-based prognostic model can accurately predict at least 5% of patients with median overall survival of less than 2 years, as well as 67% of patients with median overall survival over 5 years. Using genetic, transcriptomic, proteomic and histological analyses, we have uncovered stroma-activating pathways as well as proliferation and DNA damage response as the underlying biology of the CT-based prognostic signature. We therefore proposed treatment options accordingly to the CT-stratified patient subgroups. These results suggest a great potential for CT-images as a non-invasive, cost-effective and real-time biomarker platform in HGSOC. OPCML is a tumour suppressor gene that belongs to the IgLON protein family and is frequently silenced through promoter hyper-methylation in HGSOC. OPCML has been demonstrated to negatively regulate a panel of receptor tyrosine kinases in HGSOC and it is currently under development as a therapeutic agent. It has been suggested that other IgLON family members could be transcriptionally induced by TGFb1 and play essential functions in cancer, however, the role of OPCML in the TGFb pathway is not clear. Therefore, in the second part of this project, we investigated the transcriptional regulation and functions of OPCML in the TGFb pathway in HGSOC. We demonstrated that OPCML could be transcriptionally induced by TGFb1 in non-malignant cells via the canonical Smad pathway, while this induction is completely lost in ovarian cancer cells. Through transcriptomic and proteomic profiling, we discovered that OPCML could contribute to DNA double strand break repair, possibly via the upregulation of ATM. In summary, we have discovered novel functions of OPCML as a feedback gene in the TGFb pathway and suggested new functions for OPCML during tumorigenesis of HGSOC. With this study, we have contributed to improve the knowledge of the biology of HGSOC, and identified possible new stratification biomarkers to achieve personalised medicine in the near future.

Ovarian cancer is a heterogeneous disease, considered the most lethal of all gynaecological malignancies. With an initially high response rate to treatment, ovarian cancer can suddenly relapse, with advanced stages showing loco-regional dissemination. Despite enormous efforts to tackle ovarian cancer, current treatments fail to bring complete remission or significantly extend patient life. OPCML is a promising candidate in ovarian cancer treatment, identified as a tumour suppressor frequently inactivated in ovarian and other cancers. OPCML is a negative regulator of RTKs and regulates different cellular activities. The aims of this PhD were to identify and functionally characterise clinical OPCML mutations in ovarian cancer guided by the newly resolved crystal structure of OPCML. The project describes how TCGA and COSMIC databases have been used here to identify somatic missense mutations in several tumours. A panel of OPCML mutant constructs was then created and these selected constructs were utilised to transduce three ovarian cancer cell lines (SKOV3, PEA1, and PEO1) for domain I, and SKOV3 cells for domain II and III. Selected transduced mutants were used to characterise the mutants' localisation, interaction with receptor tyrosine kinases, proliferation, cellular transformation, invasion, migration, and their potential role in the adhesion to the extracellular matrix in vitro. Furthermore, two in vivo models were used to characterise OPCML mutants: the athymic mice model and the chick embryo chorioallantoic membrane assay. The results showed that the OPCML mutants in domain I behave differently when they interact with receptor tyrosine kinase AXL and FGFR1 compared to wild-type OPCML. These OPCML mutants preserve the ability to inhibit growth in vitro, but have a complete loss of function in vivo. They also block the OPCML inhibitory activity on anchorage-independent growth, invasion, migration, and adhesion to extracellular matrix components laminin I, fibronectin, and fibrinogen. The OPCML mutants in domain II showed partial loss of function in adhesion to collagen I and IV, and complete loss of tumour suppressor function in cell proliferation in vitro and in vivo, anchorage-independent growth, invasion, migration, and adhesion to laminin I, fibronectin, and fibrinogen. The OPCML mutants in domain III preserve adhesion to collagen I and IV, but show complete loss of function in the inhibition of tumour growth in vitro and in vivo, anchorage-independent growth, invasion, migration, and adhesion to laminin I, fibronectin, and fibrinogen. Therefore, this study identifies a new mechanism of OPCML inactivation by mutations and shows that single amino acid change can abolish OPCML function. These findings suggest that OPCML is an excellent candidate for a new and novel therapeutic strategy in ovarian cancer and other malignancies, as well as other non-cancer diseases.

Cancers from breast and ovary account for around 20% of women’s death from malignant neoplasms in the UK. Although survival from cancer has been improved, about half of cancer patients are not able to survive for more than ten years. To the date, over 600 genes have been identified to be implicated in malignant transformation and these genes can be grouped into two broad classes: oncogenes and tumour suppressor genes. In addition to the genetic or epigenetic status of these genes, aberrant intracellular trafficking of these proteins is another contributing factor that also drives deregulation in cell homeostasis. In this Ph.D. study, the intracellular trafficking of two cancer-implicated proteins was investigated: the oncogenic protein membrane type-1 matrix metalloproteinase (MT1-MMP) in breast cancer and the tumour suppressor protein opioid-binding protein cell-adhesion molecule like (OPCML) in ovarian cancer. MT1-MMP is an essential regulator of cancer cell invasion and metastasis, which are the major cause of human cancer death. The proteolytic activity of MT1-MMP promotes cellular invasion and requires this protein to be localised to invasive membrane protrusions called invadopodia. Here, we have explored the role of Rab27a in MT1-MMP secretion in breast cancer cell line MDA-MB-231. Depletion of this Rab GTPase silenced the effects of MT1-MMP-dependent gelatin degradation and 3D collagen invasion. Furthermore, Rab27a was found to be associated with MT1-MMP exocytosis-related proteins, such as IQGAP-1 and Exo84. Finally, we also showed that the regulation of MT1-MMP trafficking by Rab27a was not in biosynthetic pathway but through MT1-MMP recycling to the plasma membrane. In conclusion, we suggest that Rab27a is a novel regulator of MT1-MMP recycling in breast cancer cells. High grade serous ovarian cancer (HGSC) remains the most lethal gynaecological cancer despite advances in surgery and chemotherapy. OPCML is a glycosyl phosphatidylinositol (GPI)-anchored protein and it is downregulated in many types of cancers, including HGSC, where 84% of cases show loss of OPCML expression. Previously, our group identified that OPCML negatively regulates a specific repertoire of receptor tyrosine kinases (RTKs) by interacting with the extracellular domains of the RTKs causing a reduction in cell growth, migration and invasion. However, it has been poorly understood whether this happens on the cell surface (where OPCML is localised) or in endocytic vesicles after the two proteins are internalised together. In this study, using an ovarian cancer cell line SKOV3, we have identified that OPCML is internalised via a caveolin-1-dependent pathway, where dynamin and cholesterol were also found to be important. After the uptake, Rab5 and Rab7 appeared to be involved in the trafficking of OPCML. We could also observe active recycling and degradation of OPCML after the endocytosis. Finally, we tested the effect of OPCML on RTK distribution and trafficking. The result showed that activated AXL redistributes to caveolin-1-positive structures from clathrin-associated membranes in the presence of OPCML.

Epithelial Ovarian Cancer (EOC) is a complex and lethal gynecological malignancy as demonstrated by the heterogeneity in gene expression molecular subtypes (GEMS). To address the GEMS-specific lethality in ovarian cancer, we highlight the relevance of how a receptor tyrosine kinase (RTK) differs in the signaling pattern, functional consequences, and therapeutic implications in the context of GEMS. An enrichment analysis of the human kinome among the ovarian cancer GEMS shows that AXL is the top-ranking RTK for the EMT-driven, poor prognosis Mes subtype. Interestingly, AXL is also expressed, at a lower rank, in the epithelial, better prognosis Epi-A subtype. Upon ligand stimulation with Gas6, specific to Mes, there is recurrent temporal activation of the extracellular regulated kinase (ERK) signaling downstream of AXL. Reverse Phase Protein Analysis (RPPA) and proximity ligation assays further show that Gas6/AXL signaling transactivates other RTKs such as cMET, EGFR, and HER2 in Mes, exclusively. This RTK crosstalk in concert with the sustained activation of FRA1 downstream to ERK, coincides with the induction of the EMT driver Slug in Mes but not Epi-A. This suggests that the intrinsic property of GEMS influences the complexity and duration of signal propagation. This signal amplification downstream to the Gas6/AXL axis alludes to pathway addiction in Mes. Functionally, the recurrent ERK activation results in a motile and invasive phenotype synonymous to Mes and EMT. This further sensitizes Mes to a selective AXL inhibitor, R428, by attenuating the RTK-ERK activation, reducing the in vitro motility and invasion, and inhibiting the in ovo tumor growth in the chick chorion-allantoic membrane (CAM) assay at a lower GI50 dose compared to Epi-A. Furthermore, silencing AXL in Mes reverses the mesenchymal phenotype and abolishes tumor formation in SCID mice. Our results imply that the mesenchymal state is more sensitive to AXL inhibition, and the RTK crosstalk along the EMT gradient rewires the system, thereby drastically sensitizing it to RTK node inhibition. The epithelial state might retain a linear signaling axis and shows less therapeutic advantage to targeting AXL. Stratifying ovarian cancer patients based on the GEMS and EMT states followed by targeting AXL is promising in a prospective clinical setting. The second part of this project is modulation of AXL by the GPI-anchored tumor suppressor OPCML (Opioid-binding Protein/Cell-adhesion Molecule Like). OPCML is abrogated in 83% of sporadic EOCs and 44% of all human cancer by somatic methylation. OPCML is a potent tumor suppressor that severely attenuates tumorigenesis by down modulating a spectrum of RTKs, including, but not limited to FGFR1, FGFR3, HER2, HER4 and EPHA2. Here we show that OPCML interacts with and represses AXL. OPCML preferentially binds to the activated form of AXL and alters its membrane distribution, thereby preventing downstream oncogenic signaling and Gas6/AXL-mediated increase in migration and invasion. OPCML potentiated the effect of AXL inhibitor R428, increasing signaling inhibition and cell death. This completely novel mechanism of regulation of RTKs may be a general mechanism for coordinated regulation of signaling, and may contribute to pro-oncogenic cancer signaling through epigenetic inactivation of OPCML.