Saudi Cultural Missions Theses & Dissertations
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Item Restricted THE ROLE OF USP51 IN REGULATION OF PRC2 IN OVARIAN CANCER(University at Buffalo, 2025-05) Alghanem, Meshal; Atanassov, BoykoBackground Despite advancements in ovarian cancer treatment, resistance to platinum-based chemotherapy remains a major clinical challenge and a key contributor to disease relapse. Understanding the molecular mechanisms driving this resistance is essential for identifying new therapeutic vulnerabilities. Recent studies have shown that USP51, a deubiquitinating enzyme (DUB) involved in transcriptional regulation, is downregulated in platinum-resistant ovarian cancer cells. To further investigate the role of USP51 in epigenetic regulation in ovarian cancer cells and its role in platinum resistance, we modulated its expression via knockdown and overexpression and assessed changes in drug resistance, histone modifications, and gene expression. Methods We used shRNA-mediated knockdown and ectopic overexpression of either wild-type or catalytically inactive mutant USP51 in ovarian cancer cell lines A1847 and OVCAR5 to assess changes in histone modifications. Histone extracts and whole-cell lysates were probed by western blotting, and histone levels were quantified using ImageJ. CUT&RUN was performed to evaluate genome-wide enrichment of H2AK119ub1 and H3K27me3. RNA expression levels of target genes were analyzed using RT-qPCR. Functional assays including colony formation and MTT assays were utilized to measure the sensitivity of the ovarian cancer cells to cisplatin and EHZ2 inhibitors. Statistical significance was determined using two-tailed t-tests and two-way ANOVA with post hoc analysis. Results Our study shows that USP51 removes H2AK119ub1, a repressive histone mark added by the Polycomb Repressive Complex 1 (PRC1). This mark facilitates the recruitment of PRC2, leading to the subsequent deposition of H3K27me3, both essential for chromatin-mediated gene silencing. USP51 depletion resulted in a significant increase in global levels of both H2AK119ub1 and H3K27me3, whereas overexpression led to their reduction. These changes occurred without affecting the expression levels of PRC2 core components (EZH2, SUZ12, and EED), suggesting that USP51 acts independently of PRC2 abundance to modulate epigenetic repression. By examining genome-wide changes in chromatin states, we observed a widespread accumulation of the H2AK119ub1 and H3K27me3 marks in cells depleted of USP51. There was significant co-occupancy of these marks at shared genomic loci. Enrichment and metaplot analyses confirmed the increased deposition of these marks, supporting a model in which the loss of USP51 enhances PRC2-mediated repression. Notably, genes that were enriched for H2AK119ub1 and H3K27me3 following USP51 knockdown—such as HOXD13, NRN1, CEACAM6, and BAMBI—were significantly downregulated. In contrast, the ablation of EZH2 resulted in their overexpression. Conclusions Our results demonstrate that USP51 plays a key role in regulating gene expression in ovarian cancer cells by modulating the deposition of H3K27me3, a repressive histone mark, at target loci. Its loss results in enhanced PRC2 activity, leading to gene silencing. However, altering the levels of USP51 expression did not affect the sensitivity of cancer cells to cisplatin treatment. This implies that the reduced expression of USP51 in platinum-resistant cells is a consequence of treatment adaptation, rather than a cause. The depletion of USP51, however, increases sensitivity to EZH2 inhibitors in ovarian cancer cell lines, indicating that the loss of USP51 represents a targetable epigenetic vulnerability. Our findings establish USP51 as a crucial regulator of the chromatin landscape in ovarian cancer. This presents an opportunity for patients with low USP51 expression to benefit from PRC2-targeted therapy, which may help overcome platinum resistance and enhance treatment outcomes.35 0