Abstract 29: Functional analysis of PAX8 in high-grade serous ovarian cancer
Authors: Adler Emily K. , Lawrenson Kate , Vogiatzi Maria , Sowter Heidi , Gayther Simon 
Published: August 2012

Abstract
High-grade serous ovarian cancers (HGSOC) are the most common and most lethal ovarian cancer subtype. Identifying novel markers of HGSOC development and outcome is critical to reduce mortality. Genomic profiling of ∼500 primary HGSOCs by The Cancer Genome Atlas showed PAX8 overexpression in ∼80% of tumors. In a separate analysis of ∼350 HGSOCs, we correlated PAX8 expression with overall survival in patients. We also found significant PAX8 overexpression in 38 EOC cell lines compared to 48 normal ovarian and fallopian tube cell lines. Together, these data suggest a role for PAX8 as a frequently altered oncogene in HGSOC. To evaluate the functional significance of PAX8 in HGSOC development, we analyzed gene expression in an in vitro model of early stage neoplastic transformation, driven by CMYC and mutant KRAS expression in normal ovarian epithelial cells. PAX8 showed significantly increased expression as cells underwent stepwise transformation (p&lt;0.01). To study the putative role of PAX8 as an oncogene, we used shRNA to stably knock down gene expression in HEY HGSOC cells, and measured the in vitro transformed phenotype after knockdown. Immunofluorescence and western blotting showed a significant reduction of PAX8 expression in PAX8 knockdown cells (HEY-PAX8KD) compared to both parental cells and the scrambled shRNA control (HEY-SCR). HEY-PAX8KD cells were significantly less proliferative, and exhibited 10-fold decreased anchorage independent growth accompanied by reduced colony size. In three dimensional mass culture assays, HEY and HEY-SCR lines aggregated in smooth, dense, regularly sized spheroids; but HEY-PAX8KD cells formed irregular, diffuse spheroids incorporating only ∼50% of cells in suspension. We found no differences in Ki67 or cleaved-caspase 3 expression, suggesting differences in spheroid structure were not due to changes in proliferation or apoptosis. HEY-PAX8KD cells also showed increased migration and invasion, suggesting a novel role for PAX8 in cell-cell/cell-matrix adhesion. Differential expression of cell surface proteins between HEY and HEY-PAX8KD cells supports this hypothesis and suggests several mechanisms by which cell adhesion, migration and invasion are altered by reduced PAX8 expression. We characterized the functional significance of PAX8 in vivo, by generating multiple luciferase labeled HGSOC lines that express tetracyline-inducible PAX8 shRNAmir constructs. PAX8 inducible knockdown cells (HEYLUC-PAX8tet-KD) showed significantly decreased PAX8 expression after 48hrs of doxycycline treatment. Tumorigenicity and chemotherapeutic response with and without doxycycline treatment were evaluated after intra-peritoneal injection of HEYLUC-PAX8tet-KD cells in immunocompromised mice. Together, our data indicate that PAX8 is an essential oncogene in the development of most HGSOCs and may be a therapeutic target when combined with monoclonal antibodies against cell adhesion proteins.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 29. doi:1538-7445.AM2012-29