New strategies for identification of therapeutic target of ovarian cancer

Abstract

Ovarian cancer is the most lethal gynaecological cancer and one of the leading causes of cancer-related deaths among women worldwide, mainly due to delayed diagnosis and the high frequency of chemotherapy resistance. Carboplatin and/or paclitaxel are the first-line chemotherapy treatments in ovarian cancer. Despite the application of different treatment strategies, cancer recurrence, and the development of chemoresistance, impairs patient survival. Chemoresistance can be identified in different types of cancers, however, ovarian cancer has one of the highest rates of chemoresistance-related relapse (50% of patients, within five years). Cancer relapse typically presents as distant metastases involving other organs, where almost all second-line therapies are less effective, leading to an overall five-year survival rate of only 10–15%. Chemoresistance mechanisms can be either intrinsic or extrinsic. Intrinsic resistance results from epigenetic or phenotypic cell changes, whilst extrinsic resistance is caused by changes in the cancer cells’ microenvironment (i.e. hypoxia) or through cell–cell communication. Recent literature has identified extracellular vesicles, specifically exosomes, as important mediators of cell-cell communication. Furthermore, exosomes may serve as a minimally-invasive biomarker source to determine patient response to chemotherapy, without the need for invasive tumour biopsies. Exosomes may also provide the opportunity to enhance the effectiveness of chemotherapy.Therefore, I have investigated the role of exosomes in mediating carboplatin resistance in epithelial ovarian cancer cells. In this PhD, I aimed to characterise human epithelial ovarian cancer cell lines based on cell proliferation, migration and apoptosis (in response to carboplatin). The most aggressive cancer cell lines (higher proliferation and migration) were also resistant to carboplatin. In addition, I showed that resistant cancer cells and their corresponding exosomes were enriched with miRNAs (miR-21-5p, miR-21-3p and miR-891-5p) that are associated with chemoresistance. Transfecting sensitive cancer cells with these miRNAs increased resistance to carboplatin through the activation of several pathways involved in chemoresistance (i.e. DNA repair mechanism, efflux pump and detoxification). I also found that high levels of plasma exosomal miR-891-5p are clinically correlated with cancer recurrence in ovarian cancer patients. In a xenograft mouse model, exosomes derived from a more aggressive cell line led to significantly greater numbers of tumour nodes, compared to the less aggressive cancer cell line derived exosomes. I showed that the effect of exosomes on tumour growth and metastasis in vivo was associated with changes on the phosphorylation of β-catenin. Surprisingly, multidrug resistance protein 1 (MRP1) was high-abundant protein in tumour tissue from mice injected with SKOV-3 exosomes compared to OVCAR-3 exosomes. I then hypothesised that changes in the microenvironmental conditions (i.e. hypoxia) could change the sensitive cells’ response to chemodrugs and that the resistance phenotype (extrinsic resistance) may be transferred via exosomes. Hypoxic cells were found to be more resistant to chemodrugs and exosomes from hypoxic cells were able to transfer oncogenic molecules to parental cells under normoxic conditions, modulating the cellular response to carboplatin. This resulted in the sensitive cells becoming susceptible to acquired chemoresistance. Finally, I generated an efficient exosomal delivery system that can target ovarian cancer cells that express ephrin-B4; I engineered HEK-293T cells to express exosomes having ephrin-B2 that was fused with LAMP-2b, an exosomal membrane protein. Engineered exosomes that expressed ephrin-B2 on their surface retained their native physical properties. Targeted exosomes resulted in increased uptake by ovarian cancer tissue in vivo when compared with non-targeted exosomes. In conclusion, highly aggressive cell lines release exosomes packaged with oncogenic molecules that may act as modulators for recipient cells by inducing chemoresistance and metastases. This PhD suggests a potential role for exosomal cargo in determining patient responsiveness to a particular chemodrug. More importantly, I established a novel targeting system to improve exosomal uptake by ovarian cancer that might improve delivery of drug and gene therapies to improve the efficacy of drug delivery in the future.