Characterisation of Ovarian Cancer Development, Progression and Treatment Resistance Through Mass Spectrometry Analysis

Ovarian cancer is the deadliest gynaecological malignancy and impacts thousands of lives each year. High grade serous ovarian carcinoma (HGSOC) represents over 70% of epithelial ovarian cancers, is responsible for more than 80% of ovarian cancer related deaths and is characterised by its aggressiveness and poor prognosis. This results primarily from late detection, often after local metastasis has occurred, and a treatment response trajectory of initial response to surgery and chemotherapy, followed by relapse with chemotherapy resistant disease. Improved clinical outcomes require a detailed understanding of the molecular features which underpin ovarian cancer development, progression, and response to treatment. Through the application of mass spectrometry (MS) techniques, we provide deep and comprehensive molecular characterisation of these important features of ovarian cancer. This serves as a foundation for greater understanding of this disease with the aim of improving clinical outcomes. In pursuit of a molecular characterisation of precancerous lesions of the endometrium and fallopian tube, we applied LC-MS/MS and MALDI mass spectrometry imaging (MSI) proteomics to a rare case study exhibiting pre-cancerous lesions in the endometrium (endometrial intraepithelial carcinoma (EIC)) and fallopian tube (Serous Tubal Intraepithelial Carcinoma (STIC)) in the absence of developed cancer. Tissue from the fallopian tube was selected as studies have demonstrated that HGSOC does not develop in the ovary, but rather develops as STIC precancerous lesions in the fallopian tube. Through the development of precise sample acquisition and novel sample preparation methods, we were able to delineate between cancers and non-cancerous tissue with MSI and identify proteins in these precancerous samples, and adjacent healthy tissue. Further analysis revealed numerous metastasis associated proteins enriched in precancerous tissues compared to healthy. The development of sample preparation and MS techniques, in conjunction with identification of relevant proteomic drivers of cancer progression, provide a foundation for further molecular investigation of precancerous development of both HGSOC and endometrial cancer. The major barrier to effective treatment of ovarian cancer remains the acquired resistance to platinum-based chemotherapy, such as carboplatin (CBP). This is known to occur through various mechanisms including, but not limited to, increased drug efflux, altered cellular metabolism, altered apoptotic pathways, and improved DNA repair. However, despite this knowledge, attempts to predict chemotherapy response based on molecular features of the cancer have been unsuccessful. To address this, we applied MS analyses of proteins and metabolites to ovarian cancer cell lines and their CBP resistant pairs. Through unbiased statistical analysis we were able to separate parental from resistant cells based on their molecular profile and identify molecular and metabolic pathways which were perturbed in chemoresistant cells. However, we also identified the challenges of significant molecular heterogeneity between cell lines. This challenge was further emphasised by a proteomic MS analysis of patient derived primary cells from a chemoresistant and chemosensitive patient. The progression of ovarian cancer is facilitated by its immediate access to the peritoneal cavity which allows for cancer cells to shed from the primary tumour at an early stage and establish distant metastatic implants. To promote the survival of cancer cells in this cavity, HGSOC forms cellular aggregates within the peritoneal fluid called multicellular tumour spheroids (MCTS). These structures are resistant to chemotherapy, avoid cytoreductive surgery and are thought to represent a niche from which the cancer can re-establish itself after treatment. Further, there is significant interest in the use of an in vitro model of cancer spheroids for replicating both ovarian cancer MCTS and numerous features of solid tumours. We reviewed MS analyses of both in vitro generated MCTS and primary ovarian MCTS with the aim of understanding the molecular underpinning of their treatment response and metastatic capacity. This is concluded with a presentation of the first proteomic MALDI MSI analysis of a MCTS derived from primary patient samples in which we were able to employ spatially defined molecular features to delineate between different regions of the spheroid. There is growing interest in the use of in vitro MCTS as a model to test novel anti-cancer compounds. These structures have the advantage or replicating numerous features of solid tumours including barriers to drug penetration. Utilising MCTS, we performed a pilot study investigating the penetration of a novel CDK4/6 inhibitor (CDDD2-94) with MALDI MSI. Through this analysis we were able to clearly monitor the accumulation of the drug in different areas of the MCTS over time representing the first steps in the use of MALDI MSI to monitor drug penetration in in vitro MCTS. Distant origins, complex responses to therapy and the formation of free-floating cancer aggregates all contribute to the challenges of understanding, detecting and effectively treating ovarian cancer, and particularly HGSOC. This is reflected in the modest improvements in patient outcomes in the last 30 years, despite significant efforts by the scientific community. Through the application of advanced MS based analysis techniques, we provide comprehensive molecular information about important features in HGSOC development, treatment response and malignant progression. This sets the foundation for further research which aims to understand the early molecular events in this disease, characterise and predict chemotherapy response and advise treatment approaches which account for the molecular heterogeneity and unusual metastatic progression.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 202

A Protocol for the Acquisition of Comprehensive Proteomics Data from Single Cases Using Formalin-Fixed Paraffin Embedded Sections

The molecular analysis of small or rare patient tissue samples is challenging and often limited by available technologies and resources, such as reliable antibodies against a protein of interest. Although targeted approaches provide some insight, here, we describe the workflow of two complementary mass spectrometry approaches, which provide a more comprehensive and non-biased analysis of the molecular features of the tissue of interest. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) generates spatial intensity maps of molecular features, which can be easily correlated with histology. Additionally, liquid chromatography tandem mass spectrometry (LC-MS/MS) can identify and quantify proteins of interest from a consecutive section of the same tissue. Here, we present data from concurrent precancerous lesions from the endometrium and fallopian tube of a single patient. Using this complementary approach, we monitored the abundance of hundreds of proteins within the precancerous and neighboring healthy regions. The method described here represents a useful tool to maximize the number of molecular data acquired from small sample sizes or even from a single case. Our initial data are indicative of a migratory phenotype in these lesions and warrant further research into their malignant capabilities

Proteomic Analysis of Methylglyoxal Modifications Reveals Susceptibility of Glycolytic Enzymes to Dicarbonyl Stress

Methylglyoxal (MGO) is a highly reactive cellular metabolite that glycates lysine and arginine residues to form post-translational modifications known as advanced glycation end products. Because of their low abundance and low stoichiometry, few studies have reported their occurrence and site-specific locations in proteins. Proteomic analysis of WIL2-NS B lymphoblastoid cells in the absence and presence of exogenous MGO was conducted to investigate the extent of MGO modifications. We found over 500 MGO modified proteins, revealing an over-representation of these modifications on many glycolytic enzymes, as well as ribosomal and spliceosome proteins. Moreover, MGO modifications were observed on the active site residues of glycolytic enzymes that could alter their activity. We similarly observed modification of glycolytic enzymes across several epithelial cell lines and peripheral blood lymphocytes, with modification of fructose bisphosphate aldolase being observed in all samples. These results indicate that glycolytic proteins could be particularly prone to the formation of MGO adducts