Taxanes represent some of the most common chemotherapeutic agents for ovarian cancer treatment. However, they are only effective in approximately 40% of patients. As such, novel therapeutic strategies are required to potentiate their effect in ovarian cancer to improve patient outcome and prevent chemotherapy resistance. A hallmark of many cancers is the constitutive activation of the PI3K/AKT pathway, which drives cell survival and metabolism. In this thesis, I identified a potential vulnerability in ovarian cancer cell lines during paclitaxel-induced mitotic arrest, comprising of a striking decrease in AKT activity coupled with a significant reduction in glucose 6-phosphate and ATP levels. The reanalysis of a high content siRNA screen to discover metabolic enzymes important for ovarian cancer cell survival during paclitaxel-induced mitotic arrest, identified the metabolic enzyme PFKFB4. PFKFB4 depletion followed by paclitaxel treatment resulted in a significant decrease in mitotically arrested cells. This was accompanied by a significant increase in caspase-3/7 activity and the levels of reactive oxygen species only in mitotically arrested cells, and a significant enhancement in mitotic cell death and mitotic slippage. Depletion of the related family member, PFKFB3, demonstrated a similar phenotype. The exogenous expression of constitutively active AKT or siRNA-resistant PFKFB4 did not confer resistance to PFKFB4 depletion and paclitaxel treatment, indicating that the mechanism of mitotic cell reduction is complex. Nonetheless, the observation that some ovarian cancer cells lose AKT activity during mitotic arrest and could become vulnerable to metabolic targeting is a new concept in cancer therapy. In addition, I have identified previously unrecognised roles of PFKFB3 and PFKFB4 in mitotically arrested ovarian cancer cells. This work supports the notion that combining mitotic-targeted therapies with metabolic inhibitors may act to potentiate the effects of antimitotics in ovarian cancer cells.

Germ-line mutations within the breast cancer susceptibility genes, BRCA1 and BRCA2 are responsible for inherited susceptibility to breast and ovarian cancer. A wide spectrum of pathogenic mutations has been identified within both genes, but alterations within these genes occur far less frequently than originally believed. A large number of breast cancer families that showed linkage to BRCA1 were not found to carry a pathogenic BRCA1 mutation following the use of “classical” PCR based assays. In 1997, a large genomic rearrangement was reported within BRCA1, using Southern blotting. Numerous groups then employed semi-/quantitative methods to determine the presence and/or frequency of such alterations. This search extended the mutation spectrum of this gene, and to date at least 69 unique rearrangements have been reported. The contribution of these alterations to the burden of breast/ovarian cancer differs greatly between populations ranging from 0% to 36% of all BRCA1 mutations in the Finnish and Dutch populations respectively. Mutation screening has previously indicated that small mutations within the two BRCA genes are responsible for 59% of breast/ovarian cancer susceptibility in South Africa. To determine whether large rearrangements contribute to breast cancer susceptibility in South Africa, 74 BRCA1/2 small mutation negative patients from 58 breast / ovarian cancer families were screened for large intragenic BRCA1 rearrangements using Multiplex Ligation-dependent Probe Amplification (MLPA). In this first study of large genomic rearrangements within BRCA1 in South Africa, three genomic aberrations were detected. A deletion of exon 22 (IVS21-36del510) was identified in a Dutch immigrant. This deletion represents one of the Dutch founder mutations. Both exons 23 and 24 were found deleted in a South African family of Greek ancestry. The breakpoints of this deletion were not characterized. Simultaneous deletions of these two exons (where the breakpoints could not be characterized) have been reported in the Italian and Spanish populations. One of the genomic aberrations detected by MLPA in the present study erroneously appeared as a deletion of exon 18. Sequence analysis of this variant identified it as a single base pair substitution (c.5215G_A). This variant (R1699Q) has been reported previously, but its pathological significance is unconfirmed. In total, two large genomic rearrangements were detected in two families, of which only one is a South African, of Greek ancestry. This indicates that such mutations play a small role (1.75%; 1/57) in familial breast / ovarian cancer in South Africa (Dutch immigrant excluded). No rearrangements were identified in the Afrikaner population, indicating that such mutations do not contribute to the burden of familial breast/ovarian cancer in this population (0/40). The remaining South African breast/ovarian cancer risk may to some extent be explained by large rearrangements within BRCA2, or by mutations in other low penetrance breast cancer susceptibility gene(s). BRCA2 will now be screened by MLPA, followed by mutation screening of genes such as p53 and CHEK2 in high-risk families. / Dissertation (MSc)--University of Pretoria, 2011. / Genetics / unrestricted

Several studies have proposed the fallopian tube epithelium as a site of origin of ovarian cancer. The discovery of precursor lesions in the fallopian tube in patients at risk for ovarian cancer supports a probable origin for high-grade serous ovarian carcinoma in this tissue. While the fallopian tube epithelium consists of three distinct cell types, the paired box protein 2 (PAX2) positive cells and potentially the CD44 positive stem-like cells are most relevant to ovarian cancer. Loss of PAX2 expression in the fallopian tube cells is considered to be an early event in epithelial transformation, but the specific role of PAX2 in this transition is unknown. The aim of this study was to define the role of PAX2 in oviductal epithelial cells (OVE) cells and in mouse ovarian surface epithelial cells (MOSE), and to understand its contribution to the formation of serous precursor lesions in the fallopian tubes. Herein, we studied the OVE response to transforming growth factor β (TGFβ, a cytokine found in follicular fluid) and provide evidence of its potential involvement in the regulation of stem cell-like behaviors that may contribute to formation of cancer-initiating cells. Treatment of primary cultures of OVE cells with TGFβ at concentrations found in ovulatory follicular fluid induced an epithelial-mesenchymal transition (EMT) with expected changes in proliferation, cell morphology and expression of SNAIL, Vimentin and E-cadherin. EMT was also associated with decreased expression of PAX2 and an increase in the fraction of cells expressing CD44. Pax2 knockdown in OVE cells and overexpression in ovarian epithelial cells confirmed that PAX2 inhibits CD44 expression and regulates the degree of epithelial differentiation of OVE cells. These results suggest that the loss of PAX2 seen in serous tubal intraepithelial carcinomas (STIC) leads to a shift to a more mesenchymal phenotype associated with stem-like features. Pax2 overexpression in MOSE cells also induced the formation of vascular channels both in vitro and in vivo, which indicate a possible contribution of PAX2 to ovarian cancer progression by increasing the vascular channels to supply nutrients to the tumor cells. Furthermore, since loss of PAX2 in STIC was found associated with P53 and BRCA1 mutations, OVE cells with mutations of the tumor suppressor genes Trp53 and Brca1 were studied. We found that loss of Trp53 with or without loss of Brca1 increased cell proliferation and colony formation in vitro. In addition, loss of Trp53 induced OVE cells to undergo EMT and induced the expression of stem cell–associated genes. We therefore suggest a potential contribution of stem cells in initiating the precursor lesions in the fallopian tubes in combination with tumor suppressor gene mutation.

Philosophiae Doctor - PhD / Ovarian cancer (OC) is the most fatal gynaecologic malignancy that is generally diagnosed in the advanced stages, resulting in a low survival rate of about 40%. This emphasizes the need to identify a biomarker that can allow for accurate diagnosis at stage I. MicroRNAs (miRNAs) are appealing as biomarkers due to their stability, non-invasiveness, and differential expression in tumour tissue compared to healthy tissue. Since they are non-coding, their biological functions can be uncovered by examining their target genes and thus identifying their regulatory pathways and processes. This study aimed to identify miRNAs and genes as candidate biomarkers for early stage OC diagnosis, through two distinct in silico approaches. The first pipeline was based on sequence similarity between miRNAs with a proven mechanism in OC and miRNAs with no known role. This resulted in 9 candidate miRNAs, that have not been previously implicated in OC, that showed 90-99% similarity to a miRNA involved in OC. Following a series of in silico experimentations, it was uncovered that these miRNAs share 12 gene targets that are expressed in the ovary and also have proven implications in the disease. Since the miRNAs target genes contribute to OC onset and progression, it strengthens the notion that the miRNAs may be dysregulated as well. Using TCGA, the second pipeline involved analysing patient clinical data along with implementing statistical measures to isolate miRNAs and genes with high expression in OC. This resulted in 26 miRNAs and 25 genes being shortlisted as the potential candidates for OC management. It was also noted that targeting interactions occur between 15 miRNAs and 16 genes identified through this pipeline. In total, 35 miRNAs and 37 genes were identified from both pipelines.

In classification problems, it is always important to use the suitable combination of features that will be employed by classifiers. Generating the right combination of features usually results in good classifiers. In the situation when the problem is not well understood, data items are usually described by many features in the hope that some of these may be the relevant or most relevant ones. In this study, we focus on one such problem related to genes implicated in ovarian cancer (OC). We try to recognize two important OC-related gene groups: oncogenes, which support the development and progression of OC, and oncosuppressors, which oppose such tendencies. For this, we use the properties of promoters of these genes. We identified potential “regulatory features” that characterize OC-related oncogenes and oncosuppressors promoters. In our study, we used 211 oncogenes and 39 oncosuppressors. For these, we identified 538 characteristic sequence motifs from their promoters. Promoters are annotated by these motifs and derived feature vectors used to develop classification models. We made a comparison of a number of classification models in their ability to distinguish oncogenes from oncosuppressors. Based on 10-fold cross-validation, the resultant model was able to separate the two classes with sensitivity of 96% and specificity of 100% with the complete set of features. Moreover, we developed another recognition model where we attempted to distinguish oncogenes and oncosuppressors as one group from other OC-related genes. That model achieved accuracy of 82%. We believe that the results of this study will help in discovering other OC-related oncogenes and oncosuppressors not identified as yet.

Ovarian cancer is the second most common, and first most lethal gynecological cancer. It will affect one in seventy-eight women, and is commonly diagnosed in the later stages of the disease. The majority of the cancer’s lifespan is spent within the peritoneal cavity. Hyperthermic intraperitoneal chemotherapy (HIPEC) is an innovative new treatment that has been proven as an effective treatment in other peritoneal cancers. There is strong scientific evidence to support HIPEC as an ideal treatment for advanced ovarian cancer. Over the past two decades, there has been an increase in the number of studies focused on the efficacy of HIPEC with regards to advanced ovarian cancer. These studies have shown great promise, with two very recent phase III studies showing resounding results. It is also clear that there is a need for standardization throughout these scientific studies in order to reasonably introduce HIPEC as a standard of treatment.

Firstly, this work demonstrates that contactless dielectrophoresis (cDEP) was useful to detect a reversal in the electrical phenotype of late-stage ovarian cancer cells to a profile similar to that of slow-growing early-stage ovarian epithelial cells after treatment with a non-toxic bioactive metabolite, sphingosine. Current chemotherapeutics are highly toxic to patients and can cause severe adverse side effects, so non-toxic treatments that could slow or reverse cancer growth would be advantageous. This is the first instance of cDEP for detecting induced changes in cell structure, showing its potential as a rapid, non-biomarker-based drug screening platform. Specifically, low frequency contactless dielectrophoresis devices previously designed by Sano et al were used to extract the crossover frequency and specific membrane capacitance of early and late stage mouse ovarian surface epithelial (MOSE-E and MOSE-L) cells when untreated, treated with the anti-cancer sphingosine (So) metabolite and with a generally cancer-supporting sphingosine-1-phosphate (S1P) metabolite. The specific membrane capacitance of MOSE-L cells treated with So decreased and the normalized crossover frequency increased to levels matching MOSE-E cells. Secondly, a new multilayer cDEP device featuring curved interdigitated electrode channels overlaying a straight sample channel for the purpose of cell sorting was designed, computationally modeled, fabricated, and tested. The goal of this design was to achieve continuous multi-stream sorting of cells, and preliminary testing demonstrated that prostate cancer PC3 cells were continuously deflected toward the top of the channel under an electric field, as predicted by the numerical model. / Master of Science

No description available.

Polycystic ovarian syndrome (PCOS) is the most prevalent female endocrine disorder affecting between 5-15% of women. Characterized by a combination of polycystic ovaries, androgen excess, and abnormal ovulation, untreated PCOS may progress to metabolic abnormalities and increase the risk of adverse health outcomes. Adult PCOS is evaluated using the Rotterdam Consensus Criteria, which requires two of three clinical findings. PCOS is a condition of exclusion, and it is essential to consider differential pathologies before diagnosis. PCOS is a heterogeneous condition, and treatment is fitted to the symptoms that each individual experiences. The physiological effects of PCOS present during puberty, typically around the average age of menarche. The exact etiology of PCOS is unknown, and preventing and curing the condition is not yet possible. Metabolic disturbances caused by PCOS, including insulin resistance and increased blood glucose level, are treated with similar methods as diabetes type 2. Insulin sensitizing agents are used to treat insulin resistance caused by PCOS. The primary treatment for insulin resistance in this population is metformin (Glucophage) due to its relatively safe use and effectiveness in normalizing insulin sensitivity and assisting with normalizing weight. The correlation of PCOS with insulin resistance, central obesity, and metabolic syndrome highlights the importance of diet and exercise supplementation for this population. Weight loss of only 5% in obese and overweight PCOS patients can significantly improve PCOS symptoms, including insulin resistance, androgen levels, and fertility. Exercise alone helps increase the sensitivity of skeletal muscle to insulin and decreases metabolic syndrome risk. The effect of PCOS on the hypothalamic-pituitary-gonadal axis can be detrimental to ovulation and implantation of a fertilized egg. Treatments that suppress the HPG-axis cannot be continued during attempts to become pregnant and throughout pregnancy. Ovulation-inducing agents can improve the rate of ovulation and increase fertility; however, some women may become resistant to these treatments. Clomiphene citrate (Clomid) is often the primary drug used to induce ovulation; however, monotherapy with letrozole has shown greater improvements in pregnancy and live birth rates. Gonadotropins may also be successful treatments, but there is an accompanied increased risk of ovarian hypersensitivity syndrome and multiple pregnancies. Laparoscopic ovarian drilling may help decrease androgen production in the ovary and briefly increase pregnancy capability. During pregnancy, metformin may help decrease the risk of gestational diabetes; however, the long-term effect of fetal exposure to metformin is not well studied. Cosmetic symptoms of PCOS, including hirsutism and acne vulgaris, may cause severe social stress. PCOS women are at additional risk of depression and anxiety. Cosmetic and mental health concerns, combined with the stress caused by the high prevalence of infertility in PCOS, highlight the need for psychological help to be considered in improving the overall quality of life. Combining cognitive behavioral therapy with treatments may help PCOS women maintain treatment and improve their quality of life. The most effective treatment may require modification throughout a patient’s life due to the variance in gonadocorticoid levels throughout a female’s life. Post-menopausal women continue to have excess androgens and estrogens in circulation. High levels of ovarian and adrenal production of gonadocorticoids combined with decreased circulating binding globulins can lead to stress on the metabolic and cardiovascular systems in PCOS after menopause. Continuous levels of increased triglycerides increase the risk for atherosclerosis and adverse cardiac events. PCOS women have an increased risk of endometrial and ovarian cancer, while a link between breast cancer and PCOS is widely disputed. There is 1.66 times higher risk for cardiovascular events, including 1.96 times greater risk for stroke in women with PCOS compared to non-PCOS women when controlled for weight. As we begin to understand the increased risk factors for hypertension, hyperlipidemia, and cardiovascular stress with PCOS, it is crucial to understand how to diagnose and treat PCOS patients in the early stages of the disorder. Irregularities in typical puberty and menarche in adolescents increase the difficulty of diagnosis and may delay a diagnosis.

No description available.