Browsing by Author "Riabowol, Karl"
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Item Open Access Differential Effects of PDGFA and PDGFC Ligands on Neural Stem and Progenitor Cells(2023-12-15) Omairi, Hiba; Cairncross, John (Gregory); Goodarzi, Aaron; Riabowol, Karl; Guo, Jiami; Morris, Donald; Marra, MarcoMy thesis describes the behaviour of subventricular zone (SVZ)-derived neural stem and progenitor cells (here called NPCs) from young adult mice during exposure to members of the platelet-derived growth factor (PDGF) family of ligands. Two novel findings arise from my work. First, P53 null NPCs cultured in PDGFA transform in vitro acquiring a phenotype and genotype that closing resembles that of human glioblastoma (GBM). Second, NPCs cultured in PDGFC form small quiescent spheres of pluripotential neural stem cells (NSCs) that can be maintained in a stem-like state in PDGFC or induced to proliferate or differentiate by modifying the growth factor environment. My work is of interest to cancer biologists studying the origins of GBM and oncologists seeking ways to prevent this cancer or intercept it at an early more treatable stage. My thesis is also of interest to stem cell biologists and neurobiologists seeking new models to study the stem cell niche in the mammalian brain and brain maturation in health and disease. I have uncovered the mechanism of transformation of P53 null NPCs in PDGFA by analyzing an in vitro model of oncogenesis in which the earliest events can be detected, and their consequences tracked over time. I discovered that defective mitosis initiates a series of events that culminate in a GBM-like cancer. I also discovered that cells isolated from the SVZ using PDGFC have the behavioral and biomarker profile that would be expected of true stem cells. My findings stand in contrast to the gold standard method for isolating and studying NSCs in which cells from the SVZ are cultured in epidermal growth factor plus fibroblast growth factor (EGF/FGF) and where they divide rapidly and continuously. I also found that PDGFC could replace PDGFA in the isolation and propagation of oligodendrocyte progenitor cells (OPCs), a cell type of increasing interest. Overall, my work adds to our understanding of the biology of two important members of the PDGF family of ligands and describes new model systems that can be exploited further.Item Open Access Gender Disparities in NSCLC: A Systematic Review(2018-01-26) Alsaadoun, Noor Asaad; Bebb, Gwyn; Hollenberg, Morley; Hao, Diseree; Riabowol, Karl; Kopciuk, KarenLung cancer is the second most common malignancy in both men and women. Non-small cell lung cancer (NSCLC) represents 80-85% of cases while the remaining 15 – 20% are small cell lung cancer. Lung cancer incidence in men has steadily decreased since the mid-1980s while it has increased in women. The sex differences in smoking behavior in the last two decades partly account for this incidence pattern. Interestingly, epidemiological evidence suggests that sex alone impacts most facets of lung cancer including the incidence, susceptibility, severity, and molecular basis of the disease; however, there is lack of consensus on the etiology of the gender-based differences as well its magnitude. Therefore, we conducted an evidence-based research of the literature to identify and describe sex-associated characteristics among non-small cell lung cancer patients. We identified all potentially relevant articles published in English by searching Medline between 1996 and 2016, worldwide. Using a systematic review protocol, all abstracts were reviewed for eligibility, and relevant studies meeting inclusion criteria were retained. We included all studies on NSCLC and its main subtypes for both men and women of age over 45. Pooled data was analyzed using a semi-parametric longitudinal regression model and an ANOVA two-way test. A data-visualization tool was used to demonstrate NSCLC incidence distribution and its sex-based disparities around the world. Our data found sex-based disparities in NSCLC incidence rates, and a possible increase in female’s risk of acquiring this disease. In addition, data reveal that both race and sex have a significant effect on NSCLC incidence rates and these trends changed with time. Our findings also illustrate that global trends are not always reflective of regional ones. Results also confirms that adenocarcinoma in women is the most commonly diagnosed histology regardless of their race; however, data indicate that Asians are the dominant race to express adenocarcinomas in their lungs. The objective of this systematic literature review is to more precisely describe this gender disparity among NSCLC patients worldwide and summarize current opinions about the molecular basis for these observations. Our findings serve as a basis to begin to resolve the inherent controversies in the research, and highlight the importance of the inclusion of sex as a risk modifier in the development of screening initiatives and therapies in NSCLC.Item Open Access The ING1a Tumor Suppressor Regulates Endocytosis to Induce Cellular Senescence Via the Rb-E2F Pathway(PLoS, 2013-03-05) Rajarajacholan, Uma Karthika; Thalappilly, Subhash; Riabowol, KarlItem Embargo ING5 affects mitosis and the DNA damage response in human and murine models(2024-04-30) Estanislau Dantas, Arthur; Riabowol, Karl; Luchman, Artee; Biernaskie, Jeff; Rancourt, Derrick; Narendran, Aru; Harkness, TroyThe INhibitor of Growth family (INGs 1-5) is part of lysine acetyltransferase and lysine deacetylase complexes. They are epigenetic readers that recruit these complexes to the H3K4Me3 mark of active transcription.. Several studies have implicated ING5 in stem cell maintenance in adult and cancer stem cells, cell cycle regulation, and DNA damage response pathways. Here, we investigate the roles of ING5 in two different systems: primary mouse embryonic fibroblasts (MEFs) derived from our Ing5-KO murine model and Brain Tumor Initiating Cells (BTIC cancer stem cells) derived from glioblastoma patients. We show that Ing5-KO MEFs have defects in the cell cycle, with cells accumulating in the G2/M phase of the cell cycle, and carry a higher DNA damage load than their WT counterparts. In addition, after exposure to oxidative stress Ing5-KO cells frequently have abnormal nuclei, defective mitosis and express increased levels of the p21 cyclin-dependent kinase inhibitor compared to WT. RNA-seq analysis of Ing5-KO MEFs revealed significant downregulation of the HOXA family of transcription factors and other transcription factors involved in development such as FOXD1, SOX5 and two transcription factors of the GATA family, indicating that ING5 is part of a network for the regulation of developmental transcription factors. ING5 was also detected on the promoters of SOX4 and GATA6 in primary human fibroblasts, established human embryonic kidney (HEK293) cells and a glioma stem cell.Item Open Access Investigation of the role of ING1a in Cellular Senescence and ING1b in Ribosomal DNA transcription(2015-02-03) Rajarajacholan, Uma Karthika; Riabowol, KarlThe INhibitor of Growth (ING) proteins are type II tumor suppressors that regulate epigenetic state and transcription by functioning as stoichiometric members of histone acetyltransferase and histone deacetylase complexes. Expression of the ING1a isoform increases more than 10-fold during replicative senescence. ING1a overexpression itself induced all features of senescence examined. In this study we have identified and characterized a causal role for endocytosis and signal transduction in inducing senescence-associated phenotypes. ING1a regulated endocytosis through transcriptional activation of endocytic adaptor ITSN2. Inhibition of endocytosis altered growth factor signaling pathways leading to Rb-mediated inhibition of E2F activity, enforcing a senescence phenotype. Overexpression of ITSN2 itself induced senescence-associated characteristics in primary cells. The upstream causal role of endocytosis during senescence induction was further confirmed by ING1a-mediated increase in ITSN2 prior to the appearance of senescence markers and also by induction of senescence-associated phenotypes upon altering the stoichiometry of endocytosis regulators. ING1b isoform is well characterized in terms of regulation of cell growth and tumor suppression. Nucleolar localization of ING1b has been previously reported to be associated with stress-induced apoptosis. In this work we observe that ING1 regulates histone acetylation and the chromatin state of rDNA by recruiting HDAC1. ING1 also regulated recruitment of RNA Pol I- associated transcription factor UBF to rDNA, and affected levels of both rRNA and protein synthesis in cells. A role for ING1-mediated rRNA repression was also observed during PMA-induced differentiation of THP-1 cells. We observed that nucleolar localization of ING1 required its NTS motifs while recruitment of HDAC was dependent on the ING1 unique N-terminal region. Knock down of ING1 increased rRNA transcription and affected cellular proliferation during stress. This study identifies two novel mechanisms by which ING1 exerts its tumor suppressor function, by affecting mitogenic signaling pathways and regulating the major transcription process in cells, and confirms that epigenetic processes play an essential role in these process.Item Embargo Molecular Targeting of High-Risk Pediatric Leukemia: Identification of Targets and Biological Implications for Therapy(2024-11-26) Sharma, Ritul; Narendran, Aru; Childs, Sarah; Neri, Paola; Riabowol, KarlAcute leukemias with KMT2A (mixed-lineage leukemia or MLL) rearrangements are associated with poor prognosis and high relapse rates, particularly in infant and pediatric populations. The outcomes for patients with KMT2A-rearranged (KMT2A-r) leukemia remain suboptimal, underscoring the urgent need for novel, targeted therapeutic approaches. This thesis focuses on disrupting key molecular interactions within the KMT2A fusion protein complex that drives leukemogenesis while characterizing novel preclinical models to facilitate targeted drug discovery. By utilizing established cell line models, cell-based assays and drug combination studies, this study demonstrated the therapeutic potential of inhibiting the interaction between KMT2A fusion protein and a transcriptional kinase, CDK9. In addition, the study demonstrates that targeting CDK9 can decrease venetoclax and steroid resistance in KMT2A-r leukemia. Furthermore, this thesis investigates the biological implications of treatment induced differentiation, with a focus on identifying therapeutic strategies to treat KMT2A-r leukemias. Our results demonstrate that menin inhibitor induced differentiation in KMT2A-r cells carries a distinct inflammatory profile with elevated secretion of HMGB1 and IL-8. These cytokines are associated with promoting migratory responses. In addition, our findings showed a synergistic interaction between menin inhibition and corticosteroids, which is the first line treatment for patients with differentiation syndrome. In parallel, the study evaluates the efficacy of the ETS inhibitor TK216, targeting SPI1 overexpression in pediatric AML and B-ALL leukemias, with the goal of uncovering its therapeutic potential in high-risk pediatric leukemia. The research presented herein offers a framework for developing feasible novel therapeutic strategies for KMT2A-r leukemias, building on a foundation of preclinical modeling and mechanistic insights that may inform future clinical interventions.Item Open Access Multi-tyrosine kinase inhibitors in preclinical studies for pediatric CNS AT/RT: Evidence for synergy with Topoisomerase-I inhibition(BioMed Central, 2011-12-29) Jayanthan, Aarthi; Bernoux, Delphine; Bose, Pinaki; Riabowol, Karl; Narendran, AruItem Open Access Probing the activity of the inhibitor of growth 1a (ING1a) gene promoter under conditions of cellular stress(2024-04-30) Hill, Alexander William; Riabowol, Karl; Billon, Pierre; Williams, Gareth; Shutt, TimothyThe INhibitor of Growth (ING) family of proteins was initially characterized in 1996 when subtractive hybridization studies revealed a novel type II tumor suppressor, which the authors called ING1. Since then, additional members of the family have been discovered and characterized as important epigenetic regulators, influencing various cellular processes such as stemness, growth, and development. Previous studies have demonstrated that one of the main isoforms of ING1, named ING1a, shows increased expression in senescent cells, with overexpression of the protein resulting in the premature senescence of young fibroblasts. Classically, senescence is a phenotype that occurs as a result of telomere attrition, causing a DNA damage response that ultimately puts the cell in growth arrest. More recent work has shown that the application of various cellular stressors can induce senescence in cells which have otherwise not reached their replicative lifespan, termed stress-induced premature senescence (SIPS). Currently, the relationship between ING1a and cellular stress is incompletely understood, especially at the level of transcriptional control. In the current work, analysis of predicted transcription factors was performed for the ING1a promoter in addition to other stress and senescence-related promoters, demonstrating great overlap between ING1a and senescence marker p16. qPCR analysis of predicted factors showed decreased expression of FOXD1, FOXL1, and MAZ in senescent fibroblasts. ChIP-qPCR against MAZ was performed, validating the transcription factor prediction and showing that MAZ binds to the ING1a promoter. Comparison between promoter-driven constructs and the endogenous promoters revealed that there is likely considerable epigenetic regulation controlling the expression of ING1a, though transcriptional elements sensitive to oxidative stress are likely present.Item Open Access Regulation of Breast Cancer Cell Proliferation by APRIL(2016) Matook, Wejdan; Lee, Ki-Young; Riabowol, Karl; Brockton, NigelBreast cancer is the most common cause of death from cancer among women. “A proliferation-inducing ligand” (APRIL) is seen in the stroma of approximately 38% of breast cancer patients. APRIL is a tumor necrosis factor superfamily member that is implicated in lymphoid cell survival, proliferation and apoptosis. APRIL studies initially focused on lymphoid cells as known APRIL receptors are exclusive to these cells. However, I found that APRIL promotes breast cancer cell proliferation, and an APRIL-specific blocking peptide inhibits APRIL-induced proliferation. Therefore, I sought to identify APRIL targets in breast cancer cells. Among those identified, colony-stimulating factor 2 receptor beta (CSF2RB) is interesting. CSF2RB-APRIL interaction is direct, and the CSF2RB ligand, CSF2, has 41% amino acid sequence similarity to APRIL. CSF2RB-linked Akt and STAT3 signaling are activated in APRIL-mediated breast cancer cell proliferation. Thus, my findings raise the possibility that CSF2RB is a novel APRIL receptor in non-lymphoid cells.Item Open Access Regulation of Cell Death by The Drosophila ING Proteins(2015-05-04) Mobahat, Mahsa; Riabowol, Karl; Grewal, SavrajThe inhibitor of growth (ING) family of type II tumor suppressors (ING1-ING5) are involved in various cellular processes including apoptosis, DNA repair and tumor growth. INGs are subunits of histone deacetylase (HDAC) and histone acetyltransferase (HAT) complexes. The Drosophila genome encodes 3 ING homologues, dING 2, 3 and 4. In this research, I showed that overexpression of dING2 leads to smaller tissue and clone size in Drosophila. dING2 was sufficient to induce caspase-dependent but p53-independent cell death, which promoted expression of the pro-apoptotic gene, reaper. Experiments conducted on polyploid tissues revealed that clonal overexpression of dING2 had little effect in cells in the larval fat body, but resulted in smaller salivary glands. My experiments established that overexpression of dING3 induces caspase-dependent cell death. However, neither dING2 nor dING3 is required for IR-induced apoptosis. This work should provide valuable insights into the role of INGs in apoptosis.Item Open Access Role of ING1b in Breast Cancer Promotion(2017) Bhunia, Pritha; Riabowol, Karl; Beck, Paul; Demetrick, DougINGs or Inhibitor of Growth proteins act as type II tumor suppressors in epithelial cancers. The senescence associated secretory phenotype (SASP) is a phenomenon where senescent cells secrete a defined group of proteins including various proinflammatory cytokines. Cancer-associated fibroblasts (CAFs) are another component of the tumor microenvironment secreting various growth factors, proinflammatory cytokines and proteases. The proinflammatory cytokines secreted by CAFs and senescent cells as SASP cause chronic inflammation, which is one of the hallmarks of cancer. Although generally described as a tumor suppressor, our lab has recently reported that higher ING1 levels in the stroma correlate with poor survival outcome in breast cancer patients. To better understand this, we examined the effects of altering ING1b levels in fibroblasts. We find that ING1b increases the secretion of IL8, IL6, CXCL1 and CCL7 (also secreted by CAFs and in the SASP). This most likely occurs by ING1b activating NF-κB. Factors secreted by fibroblasts in response to ING1b also enhances the growth and migration capacity of several lines of breast cancer cells and also induce a cancer like phenotype of migration and growth in normal breast epithelial cells.Item Open Access Roles of the ING1 Epigenetic Regulator in Breast Cancer(2014-10-14) Thakur, Satbir; Riabowol, KarlING proteins are epigenetic “readers” that can target various chromatin modifying complexes to chromatin. They are involved in various cellular processes such as DNA repair, apoptosis and cellular senescence. This study focuses on examining the potential role of ING1 as a therapeutic agent and prognostic marker for breast cancer. We began by asking whether dysregulating epigenetic pathways with different chemical inhibitors could show synergistic effects with ING1 on killing cancer cells. We tested whether ING1 could synergize better with chemotherapeutics that target the same epigenetic mechanism or a different epigenetic mechanism. Combination treatment of ING1b with LBH589 (HDAC inhibitor) showed synergy, but the combination of ING1b with 5azaC (DNMT inhibitor), thus targeting two distinct epigenetic mechanisms, was more effective. Adenoviral delivery of ING1b combined with 5azaC also inhibited cancer cell growth in a xenograft model and led to tumor regression. These data showed that targeting distinct epigenetic pathways in our model was more effective in blocking cancer cell line growth than targeting the same pathway with multiple agents. Since ING1 expression is frequently repressed in breast carcinomas, but its mechanistic role in breast cancer development and metastasis was unknown, we analyzed ING1 levels in patient samples and correlated it to patient outcome. We also studied the effects of altering ING1 levels in metastasis assays in vitro and mouse metastasis model in vivo. ING1 levels were lower in tumors compared to adjacent normal breast tissue and correlated with tumor size and distant recurrence. ING1 could also predict disease-specific and distant metastasis-free survival in these patients. Decreasing levels of ING1 increased, and increasing levels decreased migration and invasion of MDA-MB231 cells in vitro. ING1 overexpression also blocked cancer cell metastasis in vivo and eliminated tumor-induced mortality in mouse models. Lastly, we determined if ING1 expression could predict breast cancer patient outcome. We found that stromal cell expression of ING1 showed an inverse correlation with patient survival. ING1 also correlated with tumor grade in these patients and multivariate analysis showed that ING1 was an independent prognostic marker in the breast cancer cohort we tested. This study provides important pre-clinical data that could help establish ING1 as a prognostic and therapeutic agent for breast cancer.Item Open Access Src Regulates the Activity of the ING1 Tumor Suppressor(PLoS, 2013-04-09) Yu, Lisa; Thakur, Satbir; Leong-Quong, Rebecca Y.Y.; Suzuki, Keiko; Pang, Andy; Bjorge, Jeffrey D.; Riabowol, Karl; Fujita, Donald J.; Fujita, Donald J.Item Open Access Structure-function analysis of ING1(2000) Scott, Michelle; Riabowol, KarlItem Open Access Survivin as a Preferential Target for Cancer Therapy(Multidisciplinary Digital Publishing Institute, 2014-02-13) Mobahat, Mahsa; Narendran, Aru; Riabowol, KarlItem Open Access The Role of the ING3 Epigenetic Regulator in Prostate Cancer(2017) Nabbi, Arash; Riabowol, Karl; Johnston, Randal; Jirik, Frank; Bismar, Tarek; Lewis, John; Morris, DonaldINhibitor of growth (ING) proteins are epigenetic regulators and stoichiometric members of histone acetyltransferase (KAT) or histone deacetylase (KDAC) complexes. By reading the histone mark H3K4me3, they direct their complexes to chromatin to alter gene expression. This thesis focuses on the role of ING3 in prostate cancer biology. Since rigorous characterization of antibodies is a prerequisite to acquire reliable results, we began by characterizing a new mouse monoclonal antibody against ING3. We profiled the expression of ING3 protein in normal human tissues and found that it is highly expressed in bone marrow, suggesting high expression in hematopoietic cell precursors. We also reported that ING3 protein levels are highest in proliferating tissues of the small intestine and epidermis. These data suggest a role for ING3 in promoting cell growth and renewal. In the second part of this study, we investigated the effects of ING3 on the androgen receptor (AR) pathway in prostate cancer (PC). We hypothesized that ING3 by virtue of being an essential member of TIP60 KAT complex, plays a role in post-translational modifications of AR protein and thereby contributes to PC progression. We found that the levels of ING3 and AR are positively correlated in patient samples and cell lines. ING3 potentiates androgen effects, activating expression of androgen responsive genes and AR-regulated reporters. We showed that ING3 interacts with the binding domain of AR and this interaction happens in the cytoplasm in the absence of androgens. ING3 increases AR-TIP60 interaction, promoting AR acetylation and nuclear translocation. The activating role of ING3 is independent of its ability to target the TIP60 complex to H3K4me3, identifying a previously unknown function for ING3. Knockdown of ING3 inhibits PC cell proliferation and migration, establishing ING3 as a positive regulator of growth in PC. Lastly, we asked whether ING3 could serve as a biomarker to distinguish latent versus aggressive PC. ING3 levels are higher in aggressive PC, with high levels of ING3 predicting shorter overall survival. Analysis with other predictive factors shows that including ING3 levels provides more accurate prognosis in PC.Item Open Access The Roles of the Smc5/6 Complex in Heterochromatin Maintenance at Telomeres(2016) Moradi Fard, Sareh; Cobb, Jennifer; Riabowol, Karl; Zaremberg, Vanina; Beattie, TaraSMC proteins constitute the core members of the Smc5/6, cohesin and condensin complexes. I demonstrate that Smc5/6 is present at telomeres throughout the cell cycle and its association with chromosome ends is dependent on Nse3, a subcomponent of the complex. Cells harboring a temperature sensitive mutant, nse3-1, are defective in Smc5/6 localization to telomeres and have slightly shorter telomeres. Shorter telomeres in nse3-1 cells correlate with a loss of Est2 association to telomeres. Nse3 interacts physically and genetically with two Rap1-binding factors, Rif2 and Sir4. When nse3-1 is combined with rif2Δ, there is a partial reversion in telomere elongation resulting from the loss of RIF2 that is independent of homologous recombination (HR). Shortening of telomeres by nse3-1 appears to be specific to rif2Δ and not a general function towards long telomeres as telomere length of nse3-1/rif1Δ cells is indistinguishable from telomere length in rif1Δ cells. Using genetic approaches, I also find that nse3-1 epistatically relates to positive regulators of telomerase, Tel1 and YKu70. Reduction in telomere-associated Smc5/6 leads to defects in telomere clustering, dispersion of the silencing factor, Sir4, and a loss in transcriptional repression for sub-telomeric genes and noncoding telomeric repeat-containing RNA (TERRA). SIR4 recovery at telomeres is reduced in cells lacking Smc5/6 functionality and vice versa. However, nse3-1/sir4Δ double mutants show additive defects for telomere shortening and TERRA regulation indicating the contribution of Smc5/6 to telomere homeostasis is only in partial overlap with SIR factor silencing. These findings support a role for Smc5/6 in telomere maintenance that go beyond its canonical role(s) in HR-mediated events during replication and telomere elongation.Item Open Access Vascular Aging: from Smooth Muscle Cell Differentiation to Polyploidization(2016) Zhan, Shi; Zheng, Xi-Long; Xilong, Zheng; Carlos, Fernandes-Patron; Bonni, Shirin; Walsh, Michael; Riabowol, KarlAging is associated with various changes in the vascular system at different structural and functional levels. These changes include increased arterial wall thickness, luminal dilation and reduced compliance. As a major component of the vascular wall, vascular smooth muscle cells (VSMCs) play an important role in exhibiting proper vascular functions. VSMCs behaviors are significantly modified by vascular aging. In this thesis, we study VSMC phenotypic modulation and polyploidiation in vascular aging through three projects. (1) Vascular aging switches VSMC phenotype from a differentiated to a dedifferentiated phenotype, which is characteristic in atherosclerosis. As an important transcription co-activator, myocardin regulates the expression of SM-differentitation marker genes and acts as a critical regulator in VSMC phenotypic modulation in response to various factors. In this thesis, we showed that GSK-3β positively regulates the expression of SM-differentiation markers in VSMCs. We also found that GSK-3β phosphorylats and enhances myocardin transcriptional activity through increasing the recruitment of myocardin to the promoter of its target gene and positively regulates myocardin gene expression. In conclusion, these results suggest that GSK-3β promotes VSMC differentiation through the regulation of myocardin activity. (2) VSMCs polyploidization is a biomarker of vascular aging. VSMC polyploidization and apoptosis co-exist in the aged or hypertensive vascular wall, but whether polyploidization contributes to VSMC apoptosis remains unknown. In this thesis, we found that nocodazole (ND), a microtubule-interfering reagent, induces VSMC polyploidization and apoptosis in a temporal order. Inhibition of ND-induced VSMC polyploidization abolished further apoptosis, suggesting a causal relatioinship between VSMC polyploidization and apoptosis. Also, we identified that mTOR signaling is involved in ND-induced VSMC polyploidization. (3) Accumulative studies have pointed a potential role of mTOR signaling in aging. To further establish the role of mTOR in VSMCs polyploidiation in aging, we established a T-REx system to overexpress mTOR in VSMCs. Overexpression of mTOR activated mTORC1 signaling and further induced VSMC polyploidization and senescence. We also found that autophagy which is negatively regulated by mTORC1 signaling is not involved in mTOR-induced VSMC polyploidy, although autohphagy has been well-established in VSMC senescence. We conclude that mTORC1 signaling induces VSMC polyploidization and senescence through different downstream effecters.