Identification and Characterization of Potential Drug Targets In Serous Ovarian Cancer
thesisposted on 2015-10-21, 00:00 authored by Eoghainin O hAinmhire
Ovarian cancer is the most lethal gynecological disease in women in the US. The Cancer Genome Atlas Network identified p53 mutations in 96% of high-grade serous ovarian carcinomas, demonstrating a critical role. Additionally, the Transforming Growth Factor Beta (TGFβ) pathway is dysfunctional in various malignancies, including ovarian cancer. This study investigated how expression of wild-type, mutant, or the absence of p53 alters ovarian cancer cell response to TGFβ signaling. It also investigated the response of the ovarian surface epithelium and the fallopian tube epithelium to TGFβ. Only ovarian cancer cells expressing wild-type p53 were growth inhibited by TGFβ, while ovarian cancer cells that were mutant or null for p53 were not growth inhibited. TGFβ induced migration in p53 null SKOV3 cells, which was not observed in SKOV3 cells with stable expression of mutant p53 (specifically the R273H mutation). Knockdown of wild-type p53 in the ovarian cancer cells, OVCA 420, enhanced cell migration in response to TGFβ. Increased protein expression of DKK1 and TMEPAI, two pro-invasive genes with enhanced expression in late stage metastatic ovarian cancer, was observed in p53 knockdown and null cells, while cells stably expressing mutant p53 demonstrated lower DKK1 and TMEPAI induction. Expression of mutant p53 or loss of p53 permit continued proliferation of ovarian cancer cell lines in the presence of TGFβ; however, cells expressing mutant p53 exhibit reduced migration as compared to wild-type and null cells, and they expressed decreased protein levels of DKK1 and TMEPAI. The cell of origin of ovarian cancer has come into question in recent years, with both the ovarian surface epithelium and the fallopian tube epithelium hypothesized to give rise to high-grade serous histotype. PAX8 is a Müllerian-expressed developmental transcription factor in the fallopian tube and high grade serous ovarian tumors, but PAX8 is not expressed in the ovarian surface epithelium. Loss of PAX8 in high-grade serous ovarian cancer leads to decreased proliferation and apoptosis. Additionally, murine models of high-grade serous ovarian cancer derived from the ovarian surface epithelium demonstrate acquired expression of PAX8 protein. This study investigated the functional and mechanistic role of PAX8 in progenitor cells of ovarian cancer. Murine OSE cells stably expressing PAX8 showed morphological and functional changes. Specifically, MOSE-PAX8 cells had increased levels of proliferation and migration, but no increase in anchorage independence. Protein and mRNA levels of pro-migratory genes, N-cadherin, fibronectin and slug, were increased in MOSE-PAX8 cells compared to control cells. Stable knockdown and knockout of PAX8 in murine oviductal epithelium could not be maintained, however, transient suppression of PAX8 did not induce cell cycle arrest or apoptosis in these cells but did slow proliferation. PAX8 is often expressed in HGSC, and several pathways associated with HGSC were shown to upregulate PAX8 regardless of the cell of origin. Pathways that augmented PAX8 expression in MOE cells include PtenshRNA, RasG12V, and combination of p53R273H with either PtenshRNA or RasG12V. Pathways that augmented PAX8 expression in MOSE cells include Lkb1shRNA, and a combination of Lkb1shRNA and PtenshRNA. demonstrating the ability of certain pathways in turning on PAX8 regardless of the cell of origin. Epithelial ovarian cancer is an aggressive disease with a high rate of therapeutic resistance, specifically chemoresistance. Unfortunately for most women, ~70% of ovarian cancers reoccur and are chemoresistant. Marine organisms have been subject to investigation in more recent times, due to the high diversity of novel compounds produced, and their use as potential therapeutics. This study investigated the potential use of aquatic actinomycete secondary metabolite production as a potential source of new therapeutics for ovarian cancer. Fraction libraries of actinomycetes collected from around the world were screened in a panel of ovarian cancer cell lines, and non-cancerous cell models representing the cells of origin of the disease. A total of 1536 fractions were screened in two ovarian cancer cell lines and two non-cancerous progenitor cells. Strain F001 produced 3 novel analogs of diazaquinomcyin (DAQ E-G), along with the known compound diazaquinomycin A (DAQA). Strain B026 produced an additional two novel analogs of diazaquinomycin. DAQA had lower LC50 values in all ovarian cancer cells compared to non-cancerous cells. In OVCAR5 cells, DAQA induced DNA damage, leading to cell cycle arrest, and eventually apoptosis. Additional fractions that showed selective cytotoxicity towards ovarian cancer cells underwent bioassay-guided fraction. Unfortunately, re-grows of the strains secreting the active compounds often led to loss of activity, or identification of already characterized molecules. However, identification of known molecules with selective toxicity demonstrated the ability of this screening system to provide a viable strategy for drug lead identification. The data obtained from the fraction library screening proves that cytotoxic compounds can be identified from aquatic actinomycetes, which may unique chemical structures and mechanisms of action.