Mervat M. Omran

Decoding Bromodomain and Extra-Terminal Domain Protein-Mediated Epigenetic Mechanisms in Human Uterine Fibroids

Uterine Fibroids (UFs) are the most common benign tumors in women of reproductive age, affecting ~77% of women overall and are clinically manifest in ~25% by age 50. Bromodomain and extra-terminal domain (BET) proteins play key roles in epigenetic transcriptional regulation, influencing many biological processes, such as proliferation, differentiation, and DNA damage response. Although BET dysregulation contributes to various diseases, their specific role in the pathogenesis of UFs remains largely unexplored. The present study aimed to determine the expression pattern of BET proteins in UFs and matched myometrium and further assess the impact of BET inhibitors on UF phenotype and epigenetic changes. Our studies demonstrated that the levels of Bromodomain-containing protein (BRD)2 and detection rate of BRD4 were significantly altered in UFs compared to matched myometrium, suggesting that aberrant BET protein expression may contribute to the pathogenesis of UFs. To investigate the biological effects of BET proteins, two small-molecule inhibitors, JQ1 and I-BET762, were used to assess their impact on UF cell behavior and transcriptomic profiles. Targeted inhibition of BET proteins markedly reduced UF cell viability compared with myometrial cells and induced cell cycle arrest. Unbiased transcriptomic profiling coupled with bioinformatic analysis revealed that BET inhibition altered multiple biological pathways, including G2M checkpoint, E2F targets, mitotic spindle, mTORC1 signaling, TNF-α signaling via NF-κB, and inflammatory response, as well as reprogrammed the UF cell epigenome. Notably, BET inhibition decreased the expression of several genes encoding extracellular matrix (ECM) proteins, a hallmark of UFs. Collectively, these results support that BET proteins play a pivotal role in regulating key signaling pathways and cellular processes in UFs. Targeting BET proteins may therefore represent a promising non-hormonal therapeutic strategy for UF treatment.

Dual Targeting EZH 2 and Histone Deacetylases in Human Uterine Sarcoma Cells Under Both 2 D and 3 D Culture Conditions

ABSTRACT Uterine sarcoma is strongly associated with poor prognosis. However, its treatment options remain limited. Tazemetostat is a potent and selective EZH2 inhibitor with limited clinical application. Entinostat is one of the strong inhibitors for HDAC1 and HDAC3. This study aimed to assess the effect of dual targeting of EZH2 and HDACs on the phenotype of uterine sarcoma cells in both 2D and 3D culture systems. The uterine sarcoma cell line (MES‐SA) was treated with varying concentrations of tazemetostat and/or entinostat for 24, 48 and 72 h. For 3D culture conditions, the cells were combined with Matrigel and seeded in V‐bottom plates and incubated for 5 days. Cell proliferation, cell cycle progression and apoptosis were evaluated. Additionally, the RNA expression, IHC staining, wound healing assay, DNMT and HDAC activity measurements were conducted. Our data showed that single‐inhibitor treatment with entinostat or tazemetostat significantly increased the cytotoxicity and significantly enhanced apoptosis concomitantly. Furthermore, both inhibitors induced cell cycle arrest in 2D and 3D culture conditions. We also demonstrated that entinostat, but not tazemetostat, suppressed the wound healing in the 2D culture. The combination treatment showed a significantly superior effect compared to single‐agent treatment. Our studies demonstrate that treatment with either entinostat or tazemetostat alone showed a potent anti‐uterine sarcoma effect in 2D and 3D culture conditions. Importantly, the combination of entinostat and tazemetostat produced superior therapeutic effects, suggesting that dual targeting EZH2 and HDACs may provide a promising treatment option for this aggressive cancer.

Utilising Human Myometrial and Uterine Fibroid Stem Cell‐Derived Three Dimentional Organoids as a Robust Model System for Understanding the Pathophysiology of Uterine Fibroids

ABSTRACTUterine fibroids (UFs) are the most common benign gynecologic tumours affecting women of reproductive age. This study aims to deepen the understanding of UFs complex aetiology through harnessing the power of 3D organoid models derived from human myometrial stem cells to emulate the in vivo behaviour of these tumours. Isolated SCs were cultured over 7 days under a defined culture system. Immunohistochemistry, Immunofluorescence, organoid stiffness, RNA Sequencing was conducted, and differential gene expression was assessed using RT‐PCR. The derived organoids exhibited diverse populations of cells, including stem cells, smooth muscle, and fibroblasts. Excessive ECM deposition was shown via Collagen and Fibronectin expression. We confirmed that our organoids expressed oestrogen receptor in a pattern similar to that in their corresponding tissue, as well as responded to steroid hormone. Interestingly, we revealed significant racial disparities in ECM accumulation within organoids derived from different racial groups. This augmented ECM deposition is theorised to enhance tissue stiffness, as assessed using Young's modulus. Additionally, our research demonstrated significant decreases in fibrotic markers upon treatment with Vitamin D3 and Doxercalciferol. Furthermore, the pro‐fibroid effects of environmental phthalates further elucidate the potential factors contributing to UF pathology. The 3D organoid model can serve as a robust platform to study the underlying molecular mechanisms of UFs, besides offering invaluable insights for potential therapeutic interventions.