Defense Date
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Biomedical Engineering
First Advisor
Barbara D. Boyan
Second Advisor
Hengbin Wang
Third Advisor
Zvi Schwartz
Fourth Advisor
Larisa Litovchick
Fifth Advisor
Priscilla Hwang
Abstract
Ovarian cancer is the deadliest gynecologic malignancy, ranking fifth in cancer-related mortality among women, with the high-grade serous ovarian carcinomas (HGSOC) being the predominant and most lethal subtype. The Remodeling and Spacing Factor 1 (RSF1) gene is frequently amplified in HGSOC, and elevated levels of RSF1 protein play a causal role in the aggressiveness and chemoresistance of HGSOC; however, the molecular mechanism remains unclear. In previous studies, we identified a ubiquitinated histone H2A (H2Aub) binding (UAB) domain in RSF1, which recognizes the H2Aub epigenetic mark. In this study, we demonstrate that recognition of H2Aub by the UAB domain is crucial for RSF1-driven ovarian cancer progression and that the interaction between H2Aub and RSF1 represents a potential target for treating ovarian cancers driven by RSF1 overexpression. Using gain-of-function approaches in SKOV3 and A2780 cells—ovarian cancer cell lines with low endogenous RSF1 levels—we showed that overexpression of RSF1, but not the UAB domain deletion mutant (RSF1ΔUAB), significantly enhanced cell proliferation, migration, invasion, and tumorigenic potential. RNA-seq analysis revealed that the interaction between RSF1 and H2Aub promoted cancer stemness and self-renewal capacity. Conversely, overexpression of a UAB domain decoy peptide reduced cell proliferation in OVCAR3 cells, an ovarian cancer cell line with high endogenous RSF1 expression. Disruption of the RSF1-H2Aub interaction in OVCAR3 cells suppressed the expression of cancer stem cell-associated genes and cell proliferation. Leveraging the specificity of UAB recognition for H2Aub nucleosomes, we developed a protocol for obtaining high-purity, homogeneous samples suitable for cryo-EM analysis, facilitating structure-based drug discovery. This approach allowed us to resolve the structure of the UAB-H2Aub nucleosome complex at a resolution of 2.6 Angstrom. Building on these studies, we are investigating the interactions between RSF1 and H2Aub nucleosomes by reconstituting the RSF1 and H2Aub nucleosome complexes for cryo-EM analysis.
In summary, the present study highlights the critical role of H2Aub recognition in RSF1-driven ovarian cancer progression and demonstrates that the interaction between RSF1 and H2Aub constitutes a druggable target for therapeutic intervention in ovarian cancers exhibiting high RSF1 expression.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
12-12-2024