Investigating the Roles of Master Cell Cycle Regulators during Cytokinesis and Embryonic Development in \u3ci\u3eCaenorhabditis elegans\u3c/i\u3e

Faithful cell division is required to maintain ploidy and generate daughter cells with necessary genetic components for life. During mitosis, dividing cells face the challenge of coordinating multiple processes to ensure that nascent daughter cells inherit an exact copy of the parent cell’s genetic identity to maintain viability. To ensure the proper execution of cell division, multiple core cell cycle proteins, such as Aurora B kinase and separase, are involved in regulating chromosome segregation, cytokinesis and abscission. Interestingly, fundamental roles for these core cell cycle proteins are being characterized in this coordination. Separase regulates chromosome segregation and vesicle trafficking during meiotic and mitotic divisions. Aurora B kinase is well characterized to eliminate incorrect attachments of kinetochore with centromere through its phosphorylation. These faultless attachments initiate a series of signaling pathways to activate separase and promote chromosome segregation. Additionally, Aurora B kinase also phosphorylates centralspindlin to complete cytokinesis and midbody formation. The collection of work presented here addresses the role of these two master cell cycle regulators in cytokinesis, abscission, and cellular events during later morphogenesis. Chapter I outlines the contribution of separase to cytokinesis, highlight how the protease activity of separase regulates exocytosis in anaphase, and suggesting that an unknown substrate is involved in separase’s regulation of exocytosis. Chapter II elucidates how programmed cytokinesis in different tissues contributes to later cellular events during morphogenesis and uncovers the novel migration pattern of midbody to apical surface. Finally, in Chapter III, we present several live imaging methods for observing C. elegans embryogenesis which were applied for this study. Collectively, the work presented here addresses the roles of these master cell cycle regulators in exocytosis, cytokinesis, abscission, and later developmental events, which is critical to understand how failure of cell division promote tumorigenesis and aneuploidy. Finally, our study may provide insightful ideas to generate clinical technologies to cure human infertility, cancer and other genetic diseases

Clinical and Translational Implications of Centrosome Amplification and Clustering in Multiple Malignancies

Cancer initiation and progression are multistep processes that rely on the generation and accumulation of non-lethal mutations, which deregulate function of tumor suppressor genes and activate oncogenic pathways. Evolving through a landscape of heterogeneous somatic mutations, mutated cells undergo subsequent selection pressures and the one endowed with the greatest fitness advantage survives giving rise to genetically diverse cell populations resulting in intratumor heterogeneity (ITH). Presence of the abnormal number of centrosomes is one of the key factors contributing towards ITH. Clustering of amplified centrosomes allows cancer cells to avoid mitotic spindle multipolarity that could otherwise result in cell death either by mitotic catastrophe or a high-grade multipolar division yielding intolerably severe aneuploidy. Thus, centrosome clustering enables low-grade chromosomal missegregation and their unequal distribution to daughter cells resulting in chromosomal instability (CIN), thus contributing to neoplastic transformation. Owing to the presence of genetically different cells in a tumor, monotargeted therapy spares clones lacking therapy-specific targets giving them the opportunity to repopulate the tumor with immunity toward the applied therapy and propensity to recur. Therefore, ITH poses major challenges to both clinicians and drug developers as it precludes detection of low-level clones, prediction of tumor evolution, development of drugs to target specific clones and evaluation of effective, yet non-toxic combinatorial regimens to combat ITH. I envision that a comprehensive quantitative analysis of centrosome amplification (CA), which is a bonafide driver of ITH might help better understand clinical behavior and improve therapeutic management of tumors. To this end, my research, presented here, primarily focuses on testing i) the impact of centrosome amplification and centrosome clustering protein (KIFC1) on clinical outcomes in multiple malignancies and ii) the role of tumor hypoxia in inducing centrosome amplification in cancer. Collectively, my findings reveal that CA and KIFC1 are prognostic and predictive in multiple malignancies and that tumor hypoxia plays a crucial role in inducing CA in tumors. This body of work expands our knowledge in causes and clinical implications of CA to help guide treatment decisions and development of precision medicine for multiple malignancies

Inducing and suppressing the alternative lengthening of telomeres mechanism in cancer cells

Cancer cells extend critically short telomeres either by reactivating the reverse transcriptase telomerase or by employing alternative lengthening of telomeres (ALT). The ALT mechanism depends on proteins involved in DNA repair and homologous recombination (HR). High-throughput sequencing is increasingly used to analyze whole genome sequences (WGS) and gene expression in patient samples, and potentially, provides a rich resource of information on ALT. However, in ALT cancers the only recurrent mutations identified so far are in the chromatin remodeler ATRX (alpha thalassemia/ mental retardation syndrome X-linked), the histone chaperone DAXX (death domain associated protein), and the histone variant H3.3. In addition, gene expression signatures for patient stratification into ALTpositive and ALT-negative as well as a systematic approach to identify genes involved in telomere maintenance (TM) and in particular ALT via functional annotation are currently missing. One wellestablished hallmark of ALT is the dislocation of the PML (promyelocytic leukemia) protein to telomeres in ALT-associated PML nuclear bodies (APBs). These colocalizations are reliable biomarkers for ALT-positive tumors, but the functional role of PML during the development of ALT remains elusive. In this thesis, I have addressed the issues raised above by work in three areas: First, the TelNet database was developed as a comprehensive compilation of TM genes. Proteins involved in TM were collected, functionally categorized, and evaluated by applying a significance score. In addition to various search modes, a statistics page was implemented for TM pathway analysis and for prediction of the active TM mechanism (TMM). Second, ALT candidate genes were identified by gene expression analysis using four different approaches and isogenic cellular systems: (i) ALT suppression by HDAC inhibitor SAHA in U2OS cells, (ii) ALT induction by ASF1 depletion in HeLa cells with long telomeres (LT), (iii) reduced ALT induction capacity of the ASF1 depletion in HeLa LT cells by SAHA treatment, and (iv) deletion of EST2 (ever shorter telomeres 2), the telomerase protein subunit, in budding yeast to generate survivors that maintain telomeres by type II recombination, equivalent to the human ALT mechanism. A differential gene expression analysis comparing perturbed cells with the unperturbed control cells revealed a positive correlation of WNT and TGFb signaling with the presence of ALT and on the other hand a negative association of TNF/ NFkB/ MAP kinase signaling. Furthermore, a role as potential ALT enhancers was predicted for KCTD15 and TNNC1. In budding yeast type II survivors, approximately 30 genes showed a relatively small albeit statistically significant change in gene expression as compared to pre-senescent cells. Genes within the iron-regulon were overrepresented among downregulated genes, including FIT1, FIT2, ARN2, and FRE4, indicating stress response. Third, I investigated the functional role of PML in the ALT pathway by recruiting PML to telomeres in cells with and without ALT background. The formation of artificial APBs induced telomere clustering and subsequently increased the abundance of extrachromosomal repeats as an ALT feature in both ALT-positive and ALT-negative cells. The results obtained in this thesis facilitate patient stratification based on deep sequencing data according to their TM mechanism and provide a better understanding of the functional role of APBs for ALT