published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Ovarian cancer is the fifth most common cause of cancer death in females and the most lethal gynecologic cancer. Globally, an estimated 240,000 people are diagnosed with ovarian cancer each year, with 22,530 new cases in the United States in 2019. Parity, oral contraceptive use, and lactation are protective, while early menarche, late menopause, and nulliparity have opposite effects. The “incessant ovulation” theory has thus emerged, in which a higher number of ovulations may be a cause of epithelial ovarian cancer (EOC). However, the mechanisms of this theory are unknown; one possibility is that the chance of acquiring a cancer-initiating pathogenic variant increases with each ovulatory cycle because of a microenvironment that promotes DNA damage. In this dissertation, we aimed to leverage genetic epidemiologic data to test this potential mechanism by evaluating the presence of gene-environment interaction between DNA repair capacity (measured through the presence of pathogenic variants in DNA repair genes) and lifetime ovulatory years (LOY). In the first aim of this dissertation, we conducted a systematic review and meta-analysis, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, to formally evaluate the strength of evidence and to generate summary point estimates for the association between LOY and EOC. We then executed two analytic aims to evaluate if the presence of pathogenic variants in DNA repair genes exacerbated the increase in ovarian cancer risk associated with LOY. In Aim 2, we evaluated interaction on the additive scale in the United Kingdom (UK) Biobank through use of a novel DNA repair capacity score developed in this dissertation, measured by quantifying the number of pathogenic variants present per individual from a list of 163 DNA repair genes, using whole exome sequencing (WES) data. In Aim 3, we evaluated the presence of interaction between pathogenic BRCA1/2 status and LOY in the Breast Cancer Family Registry (BCFR), a cohort enriched for familial risk. In both empirical aims, we assessed the presence of interaction on the additive scale using the relative excess risk due to interaction (RERI) formula. We compared results across the two empirical aims. We found the relationship between lifetime ovulations and ovarian cancer risk to be consistent and replicable in the published literature. In pooled estimates from 22 published studies, a one-year increase in LOYs was associated with a 4% (3-6%) increased risk of ovarian cancer and those with a high number of ovulations (compared to low LOYs) had a 2.15-fold (95% CI 1.82, 2.54) increased risk of ovarian cancer. We also confirmed the positive association between increasing LOYs and ovarian cancer risk in the UK Biobank and the BCFR cohorts. Although interaction on the additive scale was not detected, there were strong positive associations between pathogenic variants in DNA repair genes and ovarian cancer risk. In the UK Biobank, the presence of at least one pathogenic variant in a DNA repair gene was associated with a significant 27% increased risk of epithelial ovarian cancer (EOC) (95% CI 5-55%). Among women at high risk of ovarian cancer due to family history of breast and/or ovarian cancer, there was a strong relationship between BRCA1/2 pathogenic variants and ovarian cancer, regardless of the number of ovulations experienced. The association between LOY and ovarian cancer was found to be consistent and replicable, despite differences in study design, covariates, and measurement. We also detected robust evidence that increasing lifetime ovulations and pathogenic DNA repair variants were associated with ovarian cancer risk. Such variants were exceedingly rare in both cohorts, which limited power to detect interaction in an already rare cancer. Despite such associations, there was no evidence of synergy between LOY and impaired DNA repair capacity, but rather, high LOY and impaired DNA repair capacity may be independent risk factors of ovarian cancer. Each exposure may describe a separate class of women at increased risk of ovarian cancer that should be targeted for future prevention and screening strategies.

published_or_final_version / abstract / Obstetrics and Gynaecology / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Pathology / Master / Master of Medical Sciences

Epithelial ovarian cancer is a leading fatal malignancy in women. The high mortality rate of this cancer is due to the poor prognosis and that the majority of patients are diagnosed at late stage. Therefore, a combination of cytoreduction and adjuvant chemotherapy is the only choice for this disease at late stage. Platinum-based chemotherapy regimens are the first line treatment for ovarian cancer. However, the repetitive challenges of platinum-based agents and the frequent relapse cause acquired chemoresistance which is the major obstacle for clinical management of this disease. The underlying molecular mechanism for acquired chemoresistance remains largely unclear. Therefore, understanding the molecular mechanism in acquired chemoresistance of ovarian cancer is urgently needed. Emerging evidence has suggested that dysregulation of miRNAs is significantly involved in acquired chemoresistance in human cancers. In this study, the expression status and functional roles of miR-199b-5p were characterized in the chemoresistance of ovarian cancer. By miRCURY LNA™ miRNA array, miR-199b-5p was one of the downregulated miRNAs identified in acquired chemoresistant ovarian cancer cells. Further Q-PCR analysis found that miR-199b-5p exhibited a pattern of progressive reduction from early to advanced stage and from low to high grade ovarian cancer tissue samples (N=79). Interestingly, the expression of miR-199b-5p could be restored upon 5-Aza-2’-deoxycytidine (5-Aza-dC) treatment in ovarian cancer cells. Methylation-specific PCR and bisulfite genomic sequencing revealed that the promoter region of miR-199b-5p (Chromosome9q34) was frequently hypermethylated in chemoresistant ovarian cancer cells. Functionally, enforced expression of miR-199b-5p remarkably diminished the cisplatin-resistance in miR-199b-5p low expressing ovarian cancer cells, whereas depletion of miR-199b-5p by siRNA approach augmented cisplatin-resistance in miR-199b-5p high expressing ovarian cancer cells. A tumor xenograft mouse model further confirmed that miR-199b-5p could sensitize ovarian cancer cells to cisplatin-mediated tumor growth inhibition in vivo. On the other aspect, computational analysis plus luciferase reporter assay and western blotting identified JAG1, a key ligand of Notch1 signaling frequently associated with chemoresistance of human cancers, as a direct target of miR-199b-5p in ovarian cancer cells. Intriguingly, an inverse relationship between the downregulation of miR-199b-5p and the upregulation of JAG1 was found by in situ hybridization (ISH) and immunohistochemical (IHC) analyses in a commercial ovarian cancer tissue array (OVC1021). Functionally, enforced expression of JAG1 increased cisplatin resistance of ovarian cancer cells. In contrast, depletion of JAG1 by siRNA approach abrogated the cisplatin-resistance of ovarian cancer cells. This finding was in consistent with the effect of enforced expression of miR199b-5p in cisplatin-resistant ovarian cancer cells. Mechanistically, the Notch1 luciferase reporter assay and western blotting analysis demonstrated that the Notch1 signaling activity could be activated by JAG1 or conversely be inhibited by enforced expression of miR-199b-5p. Further investigation using the Notch specific inhibitor γ-secretase showed that JAG1-Notch1 signaling is a crucial pathway associated with chemoresistance of ovarian cancer in vitro and in vivo. Taken together, this is the first study showing that the epigenetic silencing of miR-199b-5p leads to aberrant activation of JAG1-Notch1 signaling and such signaling cascade plays a critical role in tumor progression and chemoresistance of ovarian cancer. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Ovarian cancer is one of the most deadly female malignancies. Despite advances in the treatment of ovarian cancer for the past decade, the cure rate of this disease is moderately improved. Emerging evidence suggests the molecular personalized therapeutic approach become popular for human cancer treatment. The nuclear factor-kappa B (NF-κB) signaling pathway has been shown to play multiple roles in cancer progression such as anti-apoptosis, cell cycle, angiogenesis and metastasis. This study attempted to characterize the functional roles of transforming growth factor (TGF)-β-activating kinase 1 (TAK1) in the activation of NF-κB signaling. Importantly, this study provided evidence showing the significance of TAK1-NF-κB signaling axis in ovarian cancer aggressiveness during omental metastasis. Using quantitative RT-PCR and immunohistochemical analyses, TAK1 was frequently up-regulated and was significantly associated with high-grade (P=0.001), lymph node and distant metastasis (P=0.025), as well as a tendency toward advanced stage ovarian cancers (P=0.08). Functionally, enforced expression of TAK1 could augment cell proliferation, colony formation, anchorage-independent growth ability and migration/invasion in ovarian cancer cells. Conversely, repression of TAK1 expression by genetically or pharmaceutical approach abrogated these tumorigenic capacities including tumor growth in vivo. Furthermore, co-treatment of (5Z) -7-Oxozeaenol could sensitize ovarian cancer cells to cisplatin-induced cell apoptosis, indicating TAK1 is also involved in chemoresistance. Mechanistically, using Western blotting and NF-κB -reporter luciferase analyses, the elevation of TAK1 phosphorylation at Ser412 but not Thr184/187 was found to associate with the activation of NF-κB in ovarian cancer cells solely. A series of functional studies with genetic and pharmaceutical alterations revealed that the increased TAK1 Ser412 phosphorylation was required for exerting the ovarian cancer cell oncogenesis. Omental metastasis is the common phenomenon observed in most of advanced-stage ovarian cancer. Using omentum conditioned medium (OCM), the findings of this study showed that the omentum tissue was able to secrete numerous factors including chemokines such as GRO-α and IL8 in activating TAK1-NF-κB signaling cascade, which thereby induced increased oncogenic capacities in cell growth, migration and invasion. Taken together, this study suggests that TAK1-NF-κB signaling axis is indispensable for promoting oncogenesis of ovarian cancer and targeting this pathway may be a promising personalized cancer therapeutic approach in ovarian cancer. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Heterozygous mutations in BRCA1 or BRCA2 predispose carriers to an increased risk for breast or ovarian cancer. Both BRCA1 and BRCA2 (BRCA1/2) play an integral role in promoting genomic stability through their respective actions during homologous recombination (HR) mediated repair and stalled replication fork protection from nucleolytic degradation. SMARCAD1 (SD1) is a SWI/SNF chromatin remodeler that has been implicated in promoting long-range end resection and contributes to HR. Using human cell lines, we show that SMARCAD1 promotes nucleolytic degradation in BRCA1/2-deficient cells dependent on its chromatin remodeling activity. Moreover, SMARCAD1 prevents DNA break formation and promotes fork restart at stalled replication forks. These studies identify a new role for SMARCAD1 at the replication fork. In addition to the work presented here, I discuss a method for introducing stop codons (nonsense mutations) into genes using CRISPR-mediated base editing, called iSTOP, and provide an online resource for accessing the sequence of iSTOP sgRNASs (sgSTOPs) for five base editor variants (VQR-BE3, EQR-BE3, VRER-BE3, SaBE3, and SaKKH-BE3) in humans and over 3 million targetable gene coordinates for eight eukaryotic species. Ultimately, with improvements to CRISPR base editors this method can help model and study nonsense mutations in human disease.