Determining Regions in USP15 That Regulate Its Stability and Turnover in Ovarian Cancer Cells

Due to the lack of early diagnostic markers and effective therapeutics, human ovarian cancer remains the most lethal gynecologic malignancy. This unfortunate clinical reality highlights the urgent need to identify novel therapeutic targets and more effective treatment strategies. Recent studies show that the deubiquitinase USP15 plays an important role in ovarian cancer progression and therapeutic response. Despite this, mechanisms that govern USP15 stability and turnover remain unknown. Our project aims to address this knowledge-gap by identifying regions in USP15 that regulate its stability in ovarian cancer cells. To this aim, we are cloning full-length USP15 and truncation mutants with either N- or C-terminal epitope tags into the mammalian expression vector, pcDNA3.1. Truncation mutants are generated by sequential deletion of USP15 domains, which will allow us to pin-point the regions in USP15 that are most crucial to regulating USP15 stability. Subsequently, cloned plasmids are transfected into human ovarian cancer cell lines and the half-life of the full length and truncation mutants is measured using the cycloheximide chase assay. Furthermore, a small-molecule drug called MCB-613 was previously identified to impact USP15 stability. We will also determine how MCB-613 impacts the stability of the full-length USP15 and the truncation mutants using western blot and mass spectrometry analyses. Currently, we have successfully cloned several of the USP15 mammalian expression constructs and are optimizing their transfection into ovarian cancer cell lines. The above structure-function studies will determine mechanisms and regions in USP15 that regulate USP15 stability, ultimately enabling the development of new therapeutic strategies that target USP15 in ovarian cancer.