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One of the most important risk factors in ovarian cancer is family history, and two well-studied tumour suppressor genes BRCA1 and BRCA2 have already been identified in “high-risk” families. However, alterations of other genes may also be important for ovarian cancer pathogenesis in individuals with family history of breast/ovarian cancer. In this thesis, I compared the gene expression profiles of tumours from patients with strong and weak family history of breast and/or ovarian cancer to identify genes that may be significant in the subset of patients with ovarian cancer predisposition. Based on this comparison, two genes of interest were selected for further investigations: hCDC4/FBXW7 (F-box and WD repeat domain containing 7) and PRKCZ (protein kinase C zeta). Through mutational analyses I identified one nucleotide alteration within exon 7 of hCDC4; however, overall I found that hCDC4 mutation is a rare event in ovarian tumours. Additional epigenetics analyses revealed that promoter methylation is not a significant mechanism responsible for repression of hCDC4 expression in ovarian cancer. Nevertheless, the variable expression of hCDC4 proteins observed in ovarian tumour tissues by immunohistochemical staining of tissue microarrays suggests that hCDC4 deregulation may potentially be important in a subset of ovarian cancers. Additionally, I observed that expression levels of PRKCZ are higher in ovarian tumours from patients with strong family history compared to patients with weak family history. PRKCZ has previously been shown to be involved in a variety of cellular processes; however its role in ovarian cancer remained elusive. To further understand the role of PRKCZ in ovarian tumourigenesis, including cell viability, cell migration, as well as relevant downstream signaling pathways, I performed functional assays using an in vitro ovarian cancer model. I observed that PRKCZ increases proliferation of the SKOV3 ovarian cancer cell line and participates in EGF-induced chemotaxis. Furthermore, I identified IGF1R (insulin-like growth factor 1 receptor) and ITGB3 (integrin beta 3) as downstream effectors of PRKCZ as expression of these genes is significantly altered when PRKCZ is over-expressed. Given their previously identified associations with familial ovarian cancer, the IGF1 and ITGB3 signaling pathways may therefore represent a possible link between PRKCZ and this disease.

Epithelial ovarian cancer is the leading cause of death by gynaecological malignancy. Elucidation of the driver genes of ovarian cancer will lead to treatment targets and tailored therapy for this disease. The Affymetrix Genome-Wide SNP Array 6.0 was used to study 100 serous ovarian samples and 10 normal ovarian samples to identify loci and driver genes. The ovarian cancer genome was found to have high overall genomic instability across all chromosomes and key known genes in this disease were identified in the dataset. Aberrant regions of copy number gain were located in “blocks” of constant copy number at 1p, 1q, 8q, 12p, 19q and 20q. The range in copy number for gains was 4.2 to 5.1. The “blocks” of genes were located at 8p and 5p for copy number losses. The range for copy number loss was 0.6 to 0.9.

One of the most important risk factors in ovarian cancer is family history, and two well-studied tumour suppressor genes BRCA1 and BRCA2 have already been identified in “high-risk” families. However, alterations of other genes may also be important for ovarian cancer pathogenesis in individuals with family history of breast/ovarian cancer. In this thesis, I compared the gene expression profiles of tumours from patients with strong and weak family history of breast and/or ovarian cancer to identify genes that may be significant in the subset of patients with ovarian cancer predisposition. Based on this comparison, two genes of interest were selected for further investigations: hCDC4/FBXW7 (F-box and WD repeat domain containing 7) and PRKCZ (protein kinase C zeta). Through mutational analyses I identified one nucleotide alteration within exon 7 of hCDC4; however, overall I found that hCDC4 mutation is a rare event in ovarian tumours. Additional epigenetics analyses revealed that promoter methylation is not a significant mechanism responsible for repression of hCDC4 expression in ovarian cancer. Nevertheless, the variable expression of hCDC4 proteins observed in ovarian tumour tissues by immunohistochemical staining of tissue microarrays suggests that hCDC4 deregulation may potentially be important in a subset of ovarian cancers. Additionally, I observed that expression levels of PRKCZ are higher in ovarian tumours from patients with strong family history compared to patients with weak family history. PRKCZ has previously been shown to be involved in a variety of cellular processes; however its role in ovarian cancer remained elusive. To further understand the role of PRKCZ in ovarian tumourigenesis, including cell viability, cell migration, as well as relevant downstream signaling pathways, I performed functional assays using an in vitro ovarian cancer model. I observed that PRKCZ increases proliferation of the SKOV3 ovarian cancer cell line and participates in EGF-induced chemotaxis. Furthermore, I identified IGF1R (insulin-like growth factor 1 receptor) and ITGB3 (integrin beta 3) as downstream effectors of PRKCZ as expression of these genes is significantly altered when PRKCZ is over-expressed. Given their previously identified associations with familial ovarian cancer, the IGF1 and ITGB3 signaling pathways may therefore represent a possible link between PRKCZ and this disease.

Epithelial ovarian cancer is the leading cause of death by gynaecological malignancy. Elucidation of the driver genes of ovarian cancer will lead to treatment targets and tailored therapy for this disease. The Affymetrix Genome-Wide SNP Array 6.0 was used to study 100 serous ovarian samples and 10 normal ovarian samples to identify loci and driver genes. The ovarian cancer genome was found to have high overall genomic instability across all chromosomes and key known genes in this disease were identified in the dataset. Aberrant regions of copy number gain were located in “blocks” of constant copy number at 1p, 1q, 8q, 12p, 19q and 20q. The range in copy number for gains was 4.2 to 5.1. The “blocks” of genes were located at 8p and 5p for copy number losses. The range for copy number loss was 0.6 to 0.9.

Background: High-grade serous carcinoma (HGSC) can exhibit high intratumoral heterogeneity (ITH). Despite a strong association between tumor-infiltrating lymphocytes (TIL) and survival in HGSC, ITH may have profound impacts on the anti-tumor T cell response. Yet, it is unknown how anti-tumor T cell responses contend with ITH over time in HGSC. Previous studies in melanoma and HGSC both showed tumor-reactive T cell clones emerge over time with their cognate tumor-antigens. Therefore, I hypothesized patients would share a common mechanism of T cell evolution to respond to ITH in HGSC. If so, I expect to see similar patterns of tumor recognition between primary and recurrent disease. Methods: Tumor-associated lymphocytes (TAL) were expanded from primary and recurrent ascites samples using high-dose IL-2 and a rapid-expansion protocol (REP). Following expansion, TAL were assessed for recognition of autologous tumor by IFN-γ ELISPOT and flow cytometry for CD137. CD137+ tumor-reactive TAL were FACS-purified and the tumor-reactive T cell repertoire was profiled by deep sequencing of TCRβ chains (TCRseq). Tumor-reactive TCR clonotypes were compared between primary and recurrent disease to elucidate differences in tumor-reactive populations over time in HGSC. Results: Patient TAL recognized tumor in two out of three cases. In patient IROC 060, the tumor became more immunogenic between primary and recurrent disease, which may reflect expression of new antigens and/or loss of an immunosuppressive phenotype. In patient IROC 106, the tumor remained immunogenic between primary and recurrent disease, which may reflect maintenance of stable antigen expression and an immune-sensitive phenotype. Patient IROC 034 did not exhibit any tumor-reactivity, suggesting tumor-reactivity is not ubiquitous in HGSC. FACS-purification of CD137+ T cells followed by TCRseq was successfully performed on T cell populations of both high- and low-abundance, suggesting TCRseq can be performed on populations containing very few T cells. TCRseq results that profiled the clonal repertoire of tumor-reactive TAL from primary and recurrent disease in two patients, IROC 060 and IROC 106, showed both patients had evidence of T cell loss and T cell emergence between primary and recurrent disease. Further, IROC 106 had evidence of T cell clones that were maintained between primary and recurrent disease. Conclusions: Anti-tumor T cell responses from ascites are both diverse between patients and dynamic within a patient, suggesting various mechanisms of T cell evolution to contend with ITH in HGSC. I developed a pipeline for the identification of tumor-reactive TCR sequences without the need for a priori knowledge of specific antigens. Additionally, this pipeline is feasible for very low-abundance samples, such as tumor-reactive T cells. Significance: This study provides early insights into how TAL contend with ITH in HGSC. Ultimately, these results will inform the design of adoptive T cell therapy for recurrent HGSC. / Graduate

BACKGROUND: Advanced stage epithelial ovarian cancer (EOC) presents with an extremely poor prognosis. While some patients respond well to standard treatments of chemotherapy and cytoreductive surgery, the recurrence rate is high and five-year survival is low. The importance of nutritional eating and a healthy lifestyle has been recognized as a protective factor against the development of many cancers. There may be a role for dietary and lifestyle interventions on reducing the recurrence of EOC, thereby prolonging survival. LITERATURE REVIEW FINDINGS: A review of the literature was performed, focusing on dietary and lifestyle patterns and their effects on the development and recurrence of epithelial ovarian cancers. In general, carotenoids, flavonoids, cruciferous vegetables, and flaxseed rich diets have all been proposed to reduce the risk of developing ovarian cancer. Conversely, red and processed meats, and foods with high glycemic index have been associated with an increased risk of ovarian cancer development. Additionally, obesity and high Body Mass Index (BMI) have been linked to increased cancer risk as well as increased mortality rates from cancer. PROPOSE METHODS: Given the broad spectrum of dietary and lifestyle recommendations for prevention of EOC, many providers are not aware of what information they can give to their patients regarding this topic. A Continuing Medical Education (CME) lecture summarizing the most up-to-date literature on the topic of lifestyle interventions to increase EOC survival would be beneficial. A one hour long course will be offered for CME credit to expand the knowledge base of providers and equip them with the resources necessary to educate patients on healthy lifestyle modifications in order to minimize ovarian cancer recurrence. CONCLUSIONS: With a lack of definitive cure and poor overall prognosis for women diagnosed with advanced stage epithelial ovarian cancer, there is a need for alternative treatment options when traditional methods fail. There is promising evidence that a diet high in carotenoids, cruciferous vegetables, flaxseed, and several other nutritional components is beneficial in preventing development of ovarian cancer. A diet containing processed foods, red meats, dairy, and high sugar content has been associated with increased ovarian cancer risk. It is possible that these dietary recommendations may apply to prevention of recurrence of EOC as well. The CME lecture will present the most up-to-date knowledge in the area of lifestyle interventions for the prevention of EOC. A curriculum for primary care and oncology health care providers will provide them with the tools necessary to provide their patients with nutrition and lifestyle recommendations that may prevent cancer recurrence.

OVCA is the most lethal gynecological cancer, due primarily to late diagnosis and chemoresistance (Canada, 2014; Society, 2014b). CDDP resistance is a major hurdle to successful therapy (MayoClinic, 2014). The mechanism of chemoresistance is multi-factorial including defects in apoptotic pathway and key tumor suppressor as well as dysregulation of metabolism (Borst et al., 2000; Galluzzi et al., 2012a; Siddik, 2003). Elevated aerobic glycolysis is a major source for fulfilling high energy demand of cancer, but the role of metabolic reprogramming and its regulatory mechanism in OVCA cells remain unknown. p53 is a key tumor suppressor involved in apoptosis and frequent defect of p53 (> 80%) exist in epithelial OVCA. HKII is a key metabolic enzyme involved in the first step of glycolysis and its frequent presence in the mitochondria (80% >) has been reported in multiple cancers. We demonstrate here that CDDP-induced, p53-mediated HKII down-regulation and mitochondrial p53-HKII interaction are determinants of chemosensitivity in OVCA. CDDP decreased HKII (mRNA abundance, protein level), altered its cellular localization and glycolysis in p53-wt chemosensitive OVCA cells, a response loss or attenuated in p53 deficient cells. HKII depletion sensitized chemoresistant cells to CDDP -induced apoptosis in a p53- dependent manner. In addition, p53 binds to HKII and facilitates its nuclear localization. Mechanistically, our data suggest that CDDP-activated p53 (phosphorylated p53; P-p53 Ser15) interacts with HKII in the nucleus for its regulation. Upon entry to the nucleus, P-p53(Ser15) transcriptionally regulates HKII by promoter binding, contributing to the regulation of HKII and aerobic glycolysis, eliciting apoptosis in chemosensitive OVCA cells. Conversely, this response is compromised in p53 defect chemoresistant cells. Using proximity ligation assay (PLA) in human OVCA cell lines and primary tumor cells and tumor sections from OVCA patients, we have demonstrated that nuclear HKII-P-p53(Ser15) intracellular trafficking is associated with chemosensitivity in vitro and in vivo. Furthermore, the nuclear HKII-P-p53(Ser15) interaction may be useful as a biomarker for chemosensitivity in multiple epithelial subtypes of OVCA.

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