Saudi Cultural Missions Theses & Dissertations
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Item Restricted Dissecting the Role of Enhancer of Zeste Homolog 2 (EZH2) in Neuroendocrine Transdifferentiation (NE-TD) in Non-Small Cell Lung Cancer (NSCLC) of Epithelial Origin.(Geogre Mason University, 2024-08-12) Alsubaie, Abduljalil Mohammed M; Pierobon, MariaelenaLung cancer Lung cancer is a highly prevalent and lethal disease that is responsible for a significant number of cancer-related deaths worldwide. The introduction of targeted treatments in lung cancer, like EGFR inhibitors and immunotherapies, has profoundly affected survival of NSCLC patients. Even so, the therapeutic effects of these compounds are often temporary, and resistance is regularly acquired by tumor cells to adapt to unfavorable and harmful conditions. Lineage plasticity, or the ability of cancer cells to change their physical characteristics and functions, and histological shift is emerging as important defense mechanism tumors utilize to survive treatment. For example, in response to treatment, Non-Small Cell Lung Cancers (NSCLCs) can acquire neuroendocrine (NE) characteristics and transform into Small Cell Lung Cancers (SCLCs). Conventional chemotherapy can achieve short-term response in these transformed tumors, but this mechanism of resistance remains largely untreatable and results in high levels of mortality. That is why identifying molecular drivers of NE transdifferentiation (TD) is of primary importance for devising effective therapeutic interventions and reducing lung cancer associated mortality. Overexpression of the epigenetic regulator histone-lysine N-methyltransferase Enhancer of Zeste Homolog (EZH2) has been associated with the development of SCLCs and NE-TD in prostate cancer, however, EZH2 functions has not been explored in NE transdifferentiated NSCLCs. In this study, we demonstrated that overexpression of EZH2 is associated with the acquisition of NE traits in NSCLCs and other cancers of epithelial origine. We have also demonstrated that inhibition of EZH2 in NE-TD NSCLCs reverses cancer cells to a non-NE state. Lastly, we showed that EZH2 inhibition in RB proficient tumors expressing NE markers reinstates Rb function and sensitivity to CDK4/6 inhibition. Our data provide novel understanding on the role of EZH2 in NE-TD of NSCLCs. If validated in more complex model systems, these data may provide a foundation for future clinical investigations specifically targeting patients affected by tumors for which effective therapeutic options are limited.23 0Item Restricted The Epigenetic Inhibition of EZH2 in Glioblastoma Multiforme: Identifying Potential Therapeutic Targets(Saudi Digital Library, 2023-09-18) Alrajeh, Lama; West, KatherineGlioblastoma multiforme (GBM), a highly aggressive primary brain tumor, presents significant challenges in terms of prognosis and treatment response. This study delves into the molecular intricacies of GBM, highlighting its intratumoral heterogeneity and classification into distinct subtypes with diagnostic and prognostic implications. Additionally, the role of glioma stem cells (GSCs) in GBM aggressiveness and resistance to therapy is examined, shedding light on potential therapeutic avenues. Our primary objective is to assess the effects of EZH2 inhibition, achieved through EZP and GSK inhibitors, on gene expression profiles in G7 cell lines derived from GBM. This investigation employs RNA-seq analysis to elucidate differential gene expression patterns post-EZH2 inhibition. Furthermore, we utilize ChIP-seq analysis to pinpoint direct therapeutic targets influenced by EZH2, with a focus on transcription start sites (TSS) in promoter regions. Our findings reveal a cohort of genes highly expressed in GSCs under EZH2 inhibition, including COL4A1, COL4A2, COL4A3, COL4A4, COL6A2, SYT3, BMI-1, BMP9, CHRD, NGFR, CD44, CD24, SYP, ATP1A3, ATPB2, CPE, and BCL. These discoveries provide valuable insights into the potential of EZH2 inhibition in attenuating GSCs and present promising opportunities for the development of novel therapeutic strategies in the treatment of glioblastoma.14 0Item Restricted HISTONE H3K27ME3 IN PREIMPLANTATION MOUSE EMBRYO DEVELOPMENT(Saudi Digital Library, 2023-06) Harasani, Ahlam; Pennings, Sari; Taylor, JaneDuring the developmental stages of the mouse preimplantation embryo, totipotent blastomeres generate the first three cell lineages of the embryo: trophectoderm (TE), epiblast (EPI) and primitive endoderm (PrE). The first cell fate decision consists of the separation of cells that will comprise the TE and the ICM (inner cell mass). Then, further segregation of ICM cells takes place into EPI-fated cells and PrE-fated cells. It is thought that this cell lineage specification is mediated in part by epigenetic processes, in which genes are regulated both temporally and spatially without any changes in the genome. Certain transcription factors such as Oct4, Sox2, Cdx2, Gata6, Nanog, and Fgf4 coordinate pluripotency and early cell fates during development. They are part of a network of pluripotency and associated genes that control each other’s expression as well as that of other major transcription factors through epigenetic mechanisms and simultaneous signalling pathways. Epigenetic mechanisms such as DNA methylation and post-translational histone modifications play a critical role in regulating gene expression in preimplantation embryos. During the embryonic development process, methylation of DNA is required for overall development. Similarly, trimethylation of lysine 27 on histone 3 (H3K27me3) is required to regulate developmental genes. This histone modification is facilitated by the polycomb repressive complex 2 (PRC2) subunit named enhancer of zeste homolog 1/2 (EZH1/2), an enzyme that epigenetically modulates chromatin structure and gene expression through H3K27me3 and may be involved in recruitment of DNA methyltransferases for gene silencing. The SET domain of EZH2 is thought to serve in the mechanism for transferring methyl groups from S-adenosylmethionine (SAM) to H3K27. EZH2 has a pivotal role to play in cancer progression and malignancy, but it also plays a vital role in the renewal, maintenance and differentiation of stem cells into specific cell lineages. Several recent studies have shown that blocking PRC2-EZH2 alone is not enough to suppress cancer-related genes. The dual inhibitors of both PRC2-EZH2 and PRC2-EZH1 were found to suppress H3K27me3 completely in cells after treatment. It is also suggested that Ezh1 may be involved in enhancing the maintenance of ES cell identity when EZH2 is not present. In this study, two small molecules, GSK-126 and EPZ-6438, have been used to inhibit the catalytic subunit of EZH2 in preimplantation mouse embryos in vitro to study the contribution of this enzyme through its inhibition in the embryos' developmental process. The objectives of this study are to determine the role of PRC2 complex methyltransferase EZH2 and the H3K27me3 epigenetic mark in the development of pluripotent ICM cells and other early cell fates in the mouse preimplantation embryo, based on morphology, epigenetic and cell fate markers and gene expression. This study was carried out using cultured preimplantation embryos from superovulated B6CBA F1 mice. Fertilised zygotes were collected at embryonic day 0.5 (E0.5). Embryos were cultured from age E0.5 (1-cell stage), to E1.5 (2-cell stage), E2.5 (8-cell stage) or until E4.5 (blastocyst stage) in the presence of either GSK-126 (1μM, 3μM), EPZ-6438 (0.5μM, 0.75μM, 1μM) or dimethyl sulfoxide (DMSO) as a vehicle. The effect of EZH2 inhibition was assessed based on morphology and using two techniques: reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for determining gene expression and immunocytochemistry (ICC) for measuring protein abundance. Quantitative analyses of the gene expression data of the relevant genes were conducted using the housekeeping genes GAPDH and H2Afz as endogenous controls. Indirect immunofluorescence staining was used in embryos, images were captured using confocal microscopy, and analyses were performed using MATLAB, IMARIS, and ImageJ software programs. The findings showed that inhibition of EZH2 reduced H3K27me3 levels in blastocysts. Also, the number of blastomeres was lower in the treated blastocysts than in control. EZH2 appears to influence developmental delay in the preimplantation mouse embryo. Furthermore, ICC results showed a decrease in the number of blastocyst cells expressing CDX2, the trophectoderm marker, compared with the control group. In conclusion, the results of this study will contribute to our understanding of the role H3K27me3 plays in early mammalian embryos during the specification and maintenance of cell fates.24 0