Browsing by Subject "Ovarian cancer"
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(2016)Background: Epithelial ovarian cancer is the most common type of ovarian cancer and is the most lethal gynecologic cancer due to its late diagnosis. Compared to ovarian cancer, endometrial carcinoma, as the most common gynecologic malignancy, is referred to as the “curable cancer”, as it can be detected early. As aberrant promoter methylation patterns are a common change in human cancer, detection of promoter methylation status may help in early diagnosis. In this study, we used a custom-designed methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay as a rapid and easy method, to simultaneously detect the methylation status of multiple genes in ovarian and endometrial cancer samples. Aims: To design and test an MS-MLPA assay for analyzing promoter methylation of four genes associated with ovarian and endometrial cancers. The selected genes were HNF1 homeobox B (HNF1β), Ten-eleven translocation 1(TET1), L1 cell adhesion molecule (L1CAM), and AT-rich interactive domain 1A (ARID1A). These genes are known to have expression changes by DNA methylation. Methods: The promoter DNA methylation patterns of these four genes were analyzed in 15 cancer cell lines and 5 normal cell lines and DNAs using bisulfite sequencing. Six synthetic probe pairs were designed and optimized by applying them to cancer and normal cell lines and normal DNAs and comparing the results with those of bisulfite sequencing. Finally, the MS-MLPA assay was performed on patient specimens according to the MRC-HOLLAND MS-MLPA general protocol and methylation frequencies were calculated from MS-MLPA data. Results and conclusion: The MS-MLPA assay gave accurate methylation results with the 170 samples assayed. The HNF1B, L1CAM, and TET1 Genes were observed methylated in tumor samples whereas they were not methylated in the normal samples or showed very little methylation, suggested to be favorable diagnostic markers. MS-MLPA robustly and sensitively detects the promoter DNA methylation status.
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(2019)Eph (Erythropoietin producing hepatocellular) receptors and their membrane-bound ligands, ephrins, form the largest family of receptor tyrosine kinases in mammals. They regulate functions such as cell migration and axon guidance during development, and wound healing and tissue boundary maintenance in mature tissues for example. Due to the membrane-bound nature of the ephrin ligands, Eph-ephrin signalling can proceed in two directions: forward (Eph-expressing cell) and reverse (ephrin expressing cell). In addition to its critical physiological functions in development and tissue homeostasis, the Eph-ephrin system has also been implicated in multiple diseases, including several cancers, in which the aberrant expression of Eph receptors and ephrins are often present. The EphA2 receptor for example has been identified as an oncogene with a dual mode of action as either tumor suppressor or an oncogene through distinct phosphorylation statuses of the receptor. High-grade serous ovarian cancer is the most common subtype of ovarian cancer, and the deadliest gynaecological malignancy with a dismal five-year survival rate. High-grade serous ovarian cancer does not present symptoms at an early stage, yet it quickly progresses into forming peritoneal metastases by cell shedding from the primary tumour. Small patient cohort studies have given indications of the correlation between the Eph-ephrin system and survival in ovarian cancer. This aim of this study was to investigate the impact of the Eph-ephrin system in high-grade serous ovarian cancer using a large patient cohort mRNA expression dataset to obtain survival association data of proteins of interest used with cell-based studies and the analysis of clinical samples in the form of tumor microarrays and fresh primary samples to investigate the functions of the found proteins of interest. A 428-patient The Cancer Genome Atlas high-grade serous ovarian cancer microarray mRNA dataset was analysed using the Kaplan-Meier estimator for each Eph-ephrin family member. Cell based studies were performed with recombinant ephrin treatments and ephrin knockdowns. These data were analysed using Western blotting and immunofluorescence stainings. Clinical high-grade serous ovarian cancer samples obtained from Turku University Hospital were analysed using immunohistochemistry, immunofluorescence stainings, and mRNA sequencing. EfnA5 had a significant correlation of high expression and poor survival, which is atypical to ephrins. Low EfnA3 correlated with poor survival. High levels of known oncogenes EphA2 and EphA4 also correlated with poor survival. EfnA5 treatment resulted in increased oncogenic EphA2 signaling in comparison with canonical Eph-ephrin signalling mediated by EfnA1. Knockdown of EfnA5 increased canonical, tumour suppressive EphA2 signaling, while EfnA1 knockdown increased oncogenic EphA2 signaling. Immunohistochemical analysis of tumour microarrays with multiple ovarian cancer subtypes displayed an association between the highly malignant high-grade serous subtype and EfnA5 expression. In addition to this, EfnA5 expression was increased during high-grade serous ovarian cancer progression. The Eph-ephrin system is implicated in the survival associations of multiple cancers, but the exact functions facilitated by Eph-ephrin signalling in cancer have remained relatively unknown, with the exception of EphB4-EfnB2 driven angiogenesis. This study offers insights into oncogenic Eph-ephrin signalling in ovarian cancer, displaying that oncogenic EphA2 functions can be altered by ephrins in addition to the known kinase crosstalk pathway. The noncanonical nature of EfnA5 is highlighted by its oncogenic functions in comparison to typical Eph-ephrin signalling, and the significant increase of EfnA5 expression during high-grade serous ovarian cancer progression and association with this highly malignant subtype of ovarian cancer. Although the reverse signalling effects of EfnA5 were not studied, this study highlights the importance of ephrins in Eph-ephrin signalling in cancer, presenting that the focus should not be only on the Eph receptors when studying the oncogenic signalling facilitated by the Eph-ephrin system.
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(2021)Ovarian cancers (OCs) are gynecological malignancies that cause the most gynecological cancer related deaths due to asymptomatic early-stage development and late diagnosis. The treatment of OC has not improved significantly during the last decades, and challenges are often caused by chemoresistance and the heterogeneity of cancer cell populations. Therefore, there is an urgent need to improve OC treatment outcome and implement new targeted therapies that could address the subtype specific characteristics. The most common type of OC is epithelial ovarian cancer (EOC), that can be further divided into five subtypes with distinct molecular and histological characteristics. High-grade serous subtype represents majority of cases with up to 75% of EOC patients, while other subtypes such as low-grade serous, mucinous, clear cell and endometrioid OC being less common. Considerable progress has been made in cancer treatment via precision oncology, in which individual cancer biology and tumor molecular features are investigated and used to improve treatment decisions. For this purpose, the development of patient-derived cancer cells (PDCs) offers a good opportunity to study cancer biology in vitro and to build models for preclinical molecular profiling and functional testing. PDCs can be used to establish 2D and 3D models, and most recently, wide interest has been focused on patient-derived organoids (PDOs), that offer a better model of tumor and its microenvironment, while allowing long-term culture, cryopreservation, modification and high-throughput opportunities. In this study, the aim was to establish PDO cultures using tumor cells from low-grade serous OC patients for molecular profiling and functional drug testing. PDOs were generated from both fresh and frozen tumor tissue or ascitic samples resulting to successful development of long-term PDOs from three of the five models. In order to identify optimal culturing conditions for low-grade serous OC PDOs, two previously unpublished growth mediums were tested in parallel. The more complex of the mediums showed slightly better PDO growth in general. The immunohistochemistry staining with pan-cytokeratin and PAX8 was used to confirm the epithelial and ovarian origin of PDOs. In addition, cancer panel sequencing was performed to identify mutation profiles. Importantly, the small-scale drug testing, which was performed using conventional chemotherapeutics cisplatin and paclitaxel and targeted drugs gedatolisib and trametinib, showed sample-specific responses. In conclusion, the results from this project show that PDOs are good models for ex vivo precision medicine functional studies. Importantly, we managed to establish PDOs from frozen tumor cells, suggesting that PDOs could be initiated from living biobank samples. However, the challenges related to culturing of PDOs for functional assays included slower growth rate compared to 2D cancer cell cultures and technical challenges related to Matrigel, limiting the possibilities of high-throughput drug testing. By improving these factors, PDOs will offer an efficient 3D model for preclinical use.
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(2022)Chemoresistance is a significant contributor to the lethality of high-grade serous ovarian cancer (HGSOC). Treatment response to traditional platinum-based chemotherapy is poor, and the need for improvement is urgent, as more than 50% of the patients pass within 5-years from diagnosis. Mitochondrial metabolism has emerged as a potential target in HGSOC, and enhanced capacity in mitochondrial oxidative phosphorylation (OXPHOS) has been shown to correlate with a better chemoresponse. The vital metabolic cofactor for mitochondrial enzymatic reactions, during e.g. OXPHOS, is nicotinamide adenine dinucleotide (NAD+). It is now well-established that NAD+ precursor supplementation can boost intracellular NAD+ content and, consequently, mitochondrial function. In cancer, NAD+ boosting shows mitochondrial activation mediated anticancer and chemosensitizing effects and presents an intriguing route to modulate cancer metabolism and treatment response. In HGSOC, NAD+ metabolism and its association with tumours’ metabolic profile is poorly understood. Also, the impact of mitochondrial activation on HGSOC chemoresponse remains unexplored. This thesis aimed to evaluate patient-derived HGSOC tumour NAD metabolite content and its association with OXPHOS. Also, the aim was to explore whether in vitro NAD+ boosting promotes mitochondrial function and subsequently enhances chemosensitivity to platinum-based treatment. Thus, I measured the NAD metabolite concentrations in HGSOC tumours and two HGSOC cell lines, OVCAR-5 and COV318. The impact of NAD+ boosting on HGSOC cells OXPHOS and chemoresponse was assessed with respirometry and cell viability assays. I found that the HGSOC tumours presented alterations in NAD metabolite content, with an increase in the reduced forms and a decrease in the metabolite redox ratios. Also, the change in the NAD metabolite seemed to be impacted by the tumours’ anatomical location and OXPHOS capacity. In vitro HGSOC cells differed in their OXPHOS capacity, with the OXPHOS-high cell line exhibiting enhanced sensitivity to chemotherapy. The NAD+ boosting increased intracellular NAD+ content and mitochondrial OXPHOS without impacting the cells’ chemoresponse or growth. In conclusion, the altered NAD+ metabolism in HGSOC tumours presents potential target pathways for the disease with poor treatment response. The NAD+ boosting mediated metabolic modulation increased the OXPHOS capacity independently of the cell lines’ OXPHOS-status. In OXPHOS-low cells’ mitochondrial activation enhanced OXPHOS to the level of chemosensitive OXPHOS-high cells but did not alter the cell lines’ chemoresponse within a short-term treatment period. These observations have increased the understanding of NAD+ metabolism. Also, as a proof-of-principle, NAD+ boosting was presented as a tool for mitochondrial activation and metabolic modulation in HGSOC cells, opening an intriguing approach to explore HGSOC mitochondrial function and chemoresponse.
Now showing items 1-4 of 4