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There is growing evidence that the different histological types of ovarian cancer have diverse origins. Many high-grade serous tumours (the most common type) are hypothesised to arise from the fallopian tubes, while endometrioid and clear cell tumours are hypothesised to develop from endometriosis, and the origins of mucinous tumours are uncertain. If these hypotheses are true, then the population-level risk factors for the ovarian cancer histotypes should likewise vary - but few epidemiological studies have sufficient cases to explore this. This thesis investigates the association between ovarian cancer and various exposures (including tubal ligation and reproductive factors), in a cohort of 1.3 million women, with 8,000 incident ovarian cancers. Participants completed a questionnaire at recruitment, and were followed up for routinely collected information on cancers and deaths using national registries. Cox proportional hazards models were used to estimate adjusted relative risks of ovarian cancer in women by different exposures. Tubal ligation was associated with reduced risks of cancers of the ovary, and also of the peritoneum and fallopian tube; parity and breastfeeding were also associated with reduced risks of ovarian cancer. These associations with ovarian cancer were highly heterogeneous between the different histological tumour types, consistent with hypotheses of their distinct origins. In particular, risks of endometrioid and clear cell tumours were substantially reduced amongst women with tubal ligation, but substantially increased amongst nulliparous women. For high-grade serous carcinomas, there was a smaller but significant reduction in risk with tubal ligation, and no significant association with parity. I also describe some methodological studies I set up for future tissue-based work: developing techniques in image analysis, and a pilot study of obtaining linked pathology reports and tissue samples. The population-level associations demonstrated by histotype are largely consistent with the new hypotheses of the different origins of ovarian cancer histotypes.

Ovarian cancers are the most lethal gynecological malignancies, responsible for more than 150,000 deaths around the globe annually. Among ovarian cancers, high-grade serous ovarian cancer has a 5-year survival rate of only 40%. This poor survival is due to a widespread lack of understanding of this disease, from suboptimal prevention and screening methods to failures in treatment. Moving towards novel prevention and treatment methods requires better models of ovarian cancer that phenotypically and genetically recapitulate the features of ovarian cancers that are seen clinically. This thesis highlights the characterization of a novel syngeneic model of high-grade serous ovarian cancer that exhibits the growth, expression profile, histology, and a tumor-initiating cell population that closely resembles human disease. We expand on our initial characterization of the STOSE model in a proof-of-principle study using deep learning of second-harmonic generation and two-photon-excited-fluorescence images to classify normal compared to cancerous tissues. The use of deep learning for image classification based on extracellular matrix and cellular structure could have robust application to complementing common histological examination of tissues and in treatment planning. Building on the changes in structure found in normal compared to cancerous ovarian tissue and recent research that showed age-associated fibrosis develops in murine ovaries, we assessed the non-hereditary ovarian cancer risk factors of age and ovulation number for their effects in altering ovarian tissue structure. This thesis concludes with the first evidence of ovarian fibrosis in non-pathological post-menopausal human ovaries. We show that ovarian fibrosis correlates with the development of a pre-metastatic (tumor-permissive) niche, revealing a novel avenue of research into ovarian cancer risk. Interestingly, age-associated fibrosis could be prevented or reversed by metformin use, revealing a possible mechanism for the previously identified ovarian cancer risk reduction seen with metformin use and further supporting the use of metformin for ovarian cancer prevention.

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PAX2 is a transcription factor that is essential for development. Aberrant expression of PAX2 in adult tissues is associated with carcinogenesis and experimental evidence shows that PAX2 generally exhibits oncogenic properties. Although PAX2 is not expressed in normal ovaries, it is highly expressed in low malignant potential and low-grade epithelial ovarian tumors, suggesting that PAX2 induction in ovarian surface epithelium (OSE) may contribute to transformation. Herein, we provide evidence that expression of PAX2 in normal murine OSE (mOSE) cells enhances their proliferation and survival and, when combined with loss of P53, induces tumorigenicity. In addition, OSE cells are known to gain an epithelial phenotype and express epithelial markers prior to their transformation. This study revealed that PAX2 induction in mOSE cells results in an enhanced epithelial phenotype associated with reduction of the epithelial-mesenchymal transition markers, SMA-α and COX-2. Furthermore, PAX2 inhibits the mesenchymal phenotype induced by TGF-β and reverses the TGF-β-mediated induction of both SMA-α and COX-2, in mOSE cells. Toward tumor progression, we found that when PAX2 was expressed in murine ovarian cancer cells, it enhanced or inhibited their aggressiveness, depending on the model system. In OSE cells transformed by K-RAS and MYC, PAX2 inhibited P53 accumulation and increased the level of pERK1/2 and COX-2. In addition, PAX2 inhibited apoptotic induction in these tumors, while increasing angiogenesis, both of which are enhancers of tumor aggressiveness. However, in a murine model of high-grade serous ovarian cancer, PAX2 expression reduced tumor mass and improved animal survival, likely via reduced proliferation and metastasis. Mechanistic studies showed that PAX2 increased Htra1 and decreased COX-2 in those tumors. Both HTRA1 and COX-2 are novel downstrream targets for PAX2 that are identified in the current study. These results suggest that PAX2 may not act as a classical oncogene or tumor suppressor in ovarian cancer; rather, it modulates tumorigenesis differently, depending on the tumor context. The observation that PAX2 targets distinct biological and molecular pathways might help to guide future studies to different therapeutic targets in low-grade vs. high-grade cancers.

Estrogenic hormone replacement therapy increases the risk of developing ovarian cancer, and 17-β estradiol accelerates tumour growth in mouse models of this disease. One possible mediator of estrogen action is Growth Regulation by Estrogen in Breast Cancer 1 (GREB1), an estrogen receptor alpha (ESR1)-upregulated protein. GREB1 is required for hormone-driven proliferation of several breast and prostate cancer cell lines, but its role in tumour progression is unknown and its mechanism of action remains unclear. To determine the role of GREB1 in ovarian cancer, its expression and function were examined in ovarian cancer cell lines and mouse models. GREB1 was upregulated by estradiol, and overexpression and/or knockdown of GREB1 showed that GREB1 promotes proliferation and migration in ovarian cancer cells. GREB1 knockdown also slowed tumour growth and prolonged survival in mice engrafted with ovarian cancer cells. GREB1 expression was detected in human ovarian tumours of all major histotypes, with moderate to strong expression in 75-85% of serous, endometrioid, mucinous, and clear cell carcinomas. GREB1 mRNA levels correlated with ESR1 transcripts, but protein levels of GREB1 and ESR1 did not show a correlation in tumours. Serous, endometrioid and mucinous ovarian cancers were almost always positive for either ESR1 or GREB1 (59/60 tumours), which may imply a dependence on estrogen signalling pathways. GREB1 expression in normal tissues is mainly confined to the reproductive tract, suggesting that it may be useful as a diagnostic biomarker. GREB1 combined with PAX8 showed high efficacy in differentiating mucinous tumours of gastrointestinal vs. ovarian origin. Targeting GREB1 may inhibit tumour-promoting pathways downstream of ESR1 and could therefore prove more effective than current anti-estrogen therapies.

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Ovarian cancer, primarily diagnosed at advanced stages of the disease, is the most lethal of all gynaecological malignancies, with a 5-year survival of only 45%. Increasing age and number of ovulations are the primary non-hereditary risk factors, with the median age of onset being 63 years. Considering that risk peaks upon onset of menopause and that 50% of all cases of ovarian cancer have non-ovarian origins, it is believed that the physiological aging of the ovary renders it an appealing pre-metastatic niche. Mutations in the tumor suppressor genes BRCA1 and BRCA2 are the primary hereditary risk factors, accounting for 20-25% of all cases. We have preliminary data showing that BRCA1 mutation carriers tend to develop ovarian fibrosis, a phenomenon that naturally accompanies ovarian aging, at premenopausal ages, whereas age-associated fibrosis becomes evident after menopause in non-carriers. Consistently, BRCA1/2 mutation carriers are at elevated risk for premature cessation of ovarian function. With the median age of cancer onset decreasing from 63 to 50 years of age in BRCA1 mutation carriers, these data collectively suggest accelerated ovarian aging in these women and highlights an association between ovarian aging and increased risk for cancer. As such, we hypothesized that loss of Brca1 in murine ovarian fibroblasts (MOFs) may accelerate the onset of ovarian fibrosis through fibroblast hyperactivation, contributing to ovarian aging. Using primary MOFs isolated from Brca1 LoxP/LoxP mice and adenoviral cre mediated recombination, we generated Brca1 deficient MOFs. RNA sequencing was used to characterize the transcriptomic changes associated with the loss of Brca1. Our findings suggest that Brca1 deficiency in MOFs induces cellular senescence and enhances their myofibroblastic function, likely yielding increased stiffness of the ovarian extracellular matrix due to the dysregulated synthesis and degradation of its constitutive components, contributing to accelerated ovarian aging.

Fibrinogen-like protein 2 (FGL2) expression is associated with tumour progression and poor survival in a number of different cancers. In these cancers, FGL2 promotes tumour progression via an immunosuppression-mediated mechanism or by promoting angiogenesis. Querying single-cell RNA sequencing (scRNA-seq) human cancer data shows FGL2 is produced by immune and stromal cells in the tumour microenvironment (TME), while cancer cells have minimal expression of FGL2. We therefore studied the role of FGL2 produced by cells in the TME in tumour progression in two models of cancer in which the role of FGL2 has not been previously studied: epithelial ovarian cancer (EOC) and melanoma. EOC is the most lethal gynecologic cancer with an imperative need for new treatments. Before testing novel immunotherapies, we first characterized the TME of six syngeneic ovarian cancer models. ID8-p53-/- and ID8-C3 tumours were most highly infiltrated by T cells, whereas STOSE and MOE-PTEN/KRAS tumours were primarily infiltrated by tumour associated macrophages and were unique in MHC class I and II expression. This panel of well-defined murine EOC models can serve as a valuable resource for studies that aim to test immunotherapies. We next studied the role of FGL2 in tumour progression in melanoma and ovarian cancer models. Using wild-type and FGL2 knockout mice, we found that absence of FGL2 led to a more activated TME, including activated dendritic cells (DCs - CD86+, CD40+) and T cells (CD25+, TIGIT+), as well as demonstrating for the first time that the absence of FGL2 led to more activated Natural Killer (NK) cells (DNAM-1, NKG2D) in the TME. The absence of FGL2 prolonged survival in the B16F10 model of melanoma, and synergized with oncolytic virus to prolong survival in the ID8-p53-/-Brca2-/- model of ovarian cancer.

No description available.