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Investigating the Role of PAX8 in High Grade Serous Ovarian Cancer
thesisposted on 01.05.2021, 00:00 by Laura R Hardy
Ovarian cancer is the most lethal gynecological malignancy. In 2018, there were 22,240 new cases of ovarian cancer and 14,070 deaths . The strikingly high mortality rate and lack of targeted therapies demonstrates the dire need to identify new drug targets and to develop small molecules that inhibit those targets. Development of targeted therapies, however, is hindered by the limited understanding of the molecular alterations that occur in early lesions and the ambiguity surrounding the cell of origin of ovarian cancer. Historically, the ovarian surface epithelium (OSE) was believed to be the site of ovarian cancer progenitor cells, but recent evidence suggests that the fallopian tube epithelium (FTE) is also a progenitor site. Paired box transcription factor 8 (PAX8) is a transcription factor expressed in 80-96% of high grade serous ovarian cancers (HGSOC), yet little is known about its role in tumorigenesis [2–7]. PAX8 is involved in the differentiation of Müllerian structures, including the fallopian tube and continues to be expressed in adult tissue . The ovary does not normally express PAX8, but it is acquired after malignant transformation in multiple mouse models [9,10]. Highlighting the potential importance of PAX8, an siRNA lethality screen identified PAX8 as essential for the survival of numerous ovarian cancer cell lines . In chapter two, we demonstrate that PAX8 knockdown in ovarian cancer cells led to an increase in apoptosis and a reduction in FOXM1. Conversely, inducing PAX8 expression in the OSE (MOSE-PAX8) increased proliferation, migration, and induced an epithelial-mesenchymal transition. These cells had increased expression of FOXM1, a proto-oncogene that is amplified in over 80% of HGSOC that has been implicated in several pro-cancerous functions, including invasion and migration. MOSE-PAX8 cells had increased migratory ability, and FOXM1 knockdown in these cells reversed the migratory phenotype and reduced expression of mesenchymal markers. Interestingly, silencing PAX8 in non-malignant FTE had minimal functional effects. These findings suggest PAX8 has unique roles in the OSE and FTE but regardless of cell of origin, PAX8 becomes essential in HGSOC, and is therefore an interesting drug target. In chapter three, we took a global approach using proteomic and transcriptomic methods to identify pathways regulated by PAX8 in OSE and HGSOC cell lines. The data show that PAX8 regulates cytoskeletal arrangement and increases migration and metastasis. The downstream effectors regulated by PAX8; however, are unique to each cell type. PKCa, which has previously been shown to regulate cell adhesion and migration, was upregulated by PAX8 in the OVCAR8 HGSOC cell line. Inhibition of PKC inhibited the migration of cells expressing PAX8, while activation of PKCa increased migration in cells with PAX8 deletion. Targeting PAX8 directly holds greater therapeutic promise over targeting downstream effectors because these effectors vary depending on the cell of origin, which was confirmed by showing that CRISPR deletion of PAX8 reduces tumor burden and increases survival. We demonstrate using both a genomic and pharmacological approach that PAX8 reduction is an effective therapy to attenuate HGSOC tumor progression. We also show that regardless of cell of origin, knockdown of PAX8 reduces migration. My thesis then elucidates a novel role for the small molecule thiostrepton in reducing PAX8 protein levels. Thiostrepton has previously been shown to reduce FOXM1 levels, though at high concentrations, and with significant toxicity issues. Encapsulation of thiostrepton in a sterically stabilized micelle significantly reduced tumor burden in an ovarian cancer mouse model at a lower dose than has previously been shown, and it reduced PAX8 and PKCa. These findings, taken together, suggest that PAX8 is a unifying protein driving metastasis and that micelle encapsulated thiostrepton is an effective nanomedicine to target PAX8 in HGSOC. In chapter four, we demonstrate using FTE derived syngeneic models of HGSOC, that specific oncogenic pathways alter the levels of immune cells in HGSOC progression. Our laboratory has developed cell lines derived from the fallopian tube with alterations to PTEN, KRAS, and p53 that can be used in a syngeneic system. The data demonstrate that tumors derived from the fallopian tube with PTEN shRNA KRAS G12V p53 R273H alterations have the most infiltration of both the adaptive and innate immune response. These transplantable cell line models can be used by the ovarian cancer research community to rapidly model ovarian cancer within the context of an intact immune system.