Abstract 326: Identification of novel substrates of the UBR5 E3 ubiquitin ligase in ovarian cancer

Abstract

The ubiquitin proteasome system regulates the degradation of proteins in order to maintain cellular homeostasis, which is often disrupted in cancer cells. The UBR5 (Ubiquitin Protein Ligase E3 Component N-Recognin 5) protein is an E3 ubiquitin ligase that catalyzes the direct ubiquitination of protein substrates, most often resulting in substrate protein degradation. UBR5 is the human homolog of the Drosophilia hyperplastic discs (hyd) tumor suppressor gene. However, ubr5 is amplified and over-expressed in human ovarian and breast cancers, among others, and thought to play more of a tumor-promoting role in cancer. We previously demonstrated that targeting UBR5 expression in human ovarian cancer xenografts in mice with liposomal delivery of UBR5 siRNA inhibited tumor growth and sensitized tumors to the DNA-damaging agent cisplatin. To identify novel substrates of UBR5-stimulated degradation, we transduced OVCAR5 cells with doxycycline-inducible shRNA targeting UBR5 or a control shRNA and metabolically labelled proteins with heavy or light lysine and arginine using Stable Isotope Labeling with Amino acids in Cell culture (SILAC). Doxycycline was added for the last 48 hr and cisplatin for the last 12 hrs before cell harvest. Equal amounts of protein from both cultures were mixed, digested with trypsin and analyzed by LC-MS/MS (Orbitrap Elite MS) in three independent experiments including a label swap control. Peptides and proteins were identified and quantified using MaxQuant (FDR<0.01). To identify proteins differentially expressed after UBR5 knockdown, relative protein abundance was compared by two-sided t-test with permutation-based correction of the p-value. We identified multiple proteins with increased expression following UBR5 knockdown, suggesting these proteins are regulated by ubiquitin-mediated degradation by UBR5. We validated UBR5-mediated regulation of several proteins by Western blotting, including an oncogene involved in breast and ovarian cancer progression, which we further characterized. In summary, we were able to identify several novel UBR5 candidate substrates whose expression is increased following UBR5 knockdown. Further characterization of these substrates will allow us to identify novel mechanisms of UBR5 regulation of tumor progression. Citation Format: Scott T. Eblen, Jennifer R. Bethard, D Ralph Rogers, Abdelkader Daoud, Lalima K. Madan, Lauren E. Ball. Identification of novel substrates of the UBR5 E3 ubiquitin ligase in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 326.