This collection contains theses and dissertations submitted by graduate students under the Department of Biochemistry and Molecular Biology for either a Master of Science degree or a Doctor of Philosophy degree.

Recent Submissions

  • Immune regulation of tumor cell plasticity: A promising molecular target in breast cancer metastasis

    Korkaya, Hasan; LEE, EUNMI; Department of Biochemistry and Molecular Biology / Cancer Center (2018-11-29)
    It is widely accepted that phenotypic plasticity of malignant cells is required during metastatic cascade. However, the specific mechanism of how the tumor microenvironment regulates tumor cell plasticity in metastasis is under intense investigation. We demonstrate here that monocytic and granulocytic subsets of myeloid-derived suppressor cells (MDSC), hereafter called mMDSCs and gMDSCs, infiltrate in the primary tumor and distant organs with different time kinetics and regulate spatiotemporal tumor plasticity. Using co-culture experiments and mouse transcriptome analyses in syngeneic mouse models, we provide evidence that tumor-infiltrating mMDSCs facilitate dissemination from the primary site by inducing the EMT/CSC phenotype. In contrast, pulmonary gMDSC infiltrates support metastatic growth by reverting the EMT/CSC phenotype and promoting tumor cell proliferation. We also observe that lung-derived gMDSCs isolated from tumor-bearing mice enhance metastatic growth of already disseminated tumor cells. Our ongoing studies reveal that calprotectin (S100A8 and S100A9 heterotetramer) is an important regulator of gMDSCs, which play a critical role in promoting breast cancer metastasis by inducing MET-like CSCs as well as suppressing anti-tumor immunity within the pre-metastatic niche. Furthermore, we develop a novel gMDSC-targeting compound that potentially binds to calprotectin and validate its therapeutic utility in a preclinical breast cancer model. Our goal for this study is to elucidate the molecular co-evolution of tumor and immune cells in cancer development and to identify molecular targets to provide alternative therapeutic options for women with metastatic disease.
  • Canonical Wnt Signaling in Antigen Presenting Cells Regulates Microbiota-Induced Inflammation and Immune Cell Homeostasis in the Colon

    Swafford, Daniel Joseph; Department of Biochemistry and Molecular Biology / Cancer Center (8/3/2018)
    Aberrant Wnt/β-catenin-signaling occurs in several inflammatory diseases including inflammatory bowel disease (IBD) and IBD-associated colon carcinogenesis. However, its role in shaping mucosal immune responses to commensals in the gut remains unknown. Here, we investigated the importance of canonical Wnt signaling in CD11c+ antigen presenting cells (APCs) in controlling intestinal inflammation. Using a mouse model of ulcerative colitis, we demonstrated that canonical Wnt-signaling in intestinal CD11c+ antigen presenting cells (APCs) controls intestinal inflammation by imparting an anti-inflammatory phenotype. Genetic deletion of Wnt co-receptors, low-density lipoprotein receptor-related protein 5 and 6 (LRP5/6) in CD11c+ APCs in mice (LRP5/6ΔCD11c mice) resulted in enhanced intestinal inflammation with increased histopathological severity of colonic tissue. This was due to microbiota-dependent increased production of pro-inflammatory cytokines and decreased expression of immune regulatory factors such as IL-10, retinoic acid (RA), and IDO. In addition, loss of LRP5/6-mediated signaling in CD11c+ APCs resulted in altered microflora and T cell homeostasis, which led to a loss of systemic tolerance to oral antigen. Furthermore, our study demonstrates that conditional activation of β-catenin in CD11c+ APCs in LRP5/6ΔCD11c mice resulted in reduced acute intestinal inflammation with decreased histopathological severity of colonic tissue. Loss of canonical Wnt signaling in CD11c+ APCs also results in an increase in colonic polyp formation and exacerbation of chronic inflammation/injury. This was also heavily dependent on the presence and composition of the gut microbiota, as fecal transfers from LRP5/6ΔCD11c mice to floxed control (LRP5/6FL/FL) mice that were administered an antibiotic cocktail produces a polyp load and weight loss similar to that of LRP5/6ΔCD11c mice without treatment. Additionally, our study demonstrates that conditional activation of β-catenin in CD11c+ APCs in LRP5/6ΔCD11c mice reduces severity of inflammation-associated colon carcinogenesis in these mice. Furthermore, we show that treatment of LRP5/6ΔCD11c mice with either RA or IL-10 reduces severity of inflammation-associated colon carcinogenesis. Mechanistically, RA and IL-10 may independently reduce key inflammatory factors at the acute phase of colitis. These results ultimately reveal a mechanism by which intestinal APCs control intestinal inflammation and immune homeostasis via the canonical Wnt signaling pathway, which may serve as a promising target for chronic inflammatory disorders.

    Arbab, Ali S.; Angara, Kartik Prasad; Department of Biochemistry and Molecular Biology (7/20/2018)
    Glioblastoma (GBM) is a hypervascular and hypoxic neoplasia of the central nervous system with an extremely high rate of mortality. Owing to its hypervascularity, anti-angiogenic therapies (AAT) have been used as an adjuvant to the traditional surgical resection, chemotherapy, and radiation to normalize blood vessels, control abnormal vasculatures and prevent recurrence. The benefits of AAT have been transient and the tumors were shown to relapse faster and demonstrated particularly high rates of AAT-induced therapy resistance due to activation of alternative neovascularization mechanisms. Vascular Mimicry (VM) is the uncanny ability of tumor cells to acquire endothelial-like properties, lay down vascular patterned networks reminiscent of host endothelial blood vessels and served as an irrigation system for the tumors to meet with the increasing metabolic and nutrient demands in the event of the ensuing hypoxia resulting from AAT. In our studies, we have demonstrated that AAT accelerates VM. We observed that Vatalanib (a VEGFR2 tyrosine kinase inhibitor) induced VM vessels are positive for periodic acid-Schiff (PAS) matrix but devoid of any endothelium on the inner side and lined by tumor cells on the outer side. Interestingly, 20-HETE synthesis inhibitor HET0016 significantly decreased GBM tumors through decreasing VM structures both at the core and at the periphery of the tumors. During our extensive studies to understand the tumor-inherent mechanisms of AAT-induced resistance, we identified a crucial chemokine, CXCL8 or IL-8, to be highly upregulated in the GBM tumors treated with AAT. IL-8 has been well established as a highly prevalent cytokine in GBM with potent pro-migratory and pro-angiogenic functions. AAT-treated groups had significantly higher populations of CXCR2+ glioma stem cells and endothelial-like subpopulations and these populations were decreased following treatment with HET0016 and SB225002 (a CXCR2 antagonist). CXCR2+ GBM tumor cells were shown to form VM-like vascular channels carrying functional RBCs. Knocking down CXCR2 led to smaller tumor size in the animals and improperly developed vascular structures without CXCR2+ GBM cells lining them. This confirms our hypothesis that CXCR2+ GBM cells initiate VM and contribute to AAT resistance in GBM. Our present study suggests that HET0016 and SB225002 have potential to target therapeutic resistance and can be combined with other antitumor agents in preclinical and clinical trials.

    Elmasry, Khaled; Department of Biochemistry and Molecular Biology / Cancer Center (5/22/2018)
    Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO-induced retinal changes in diabetic retinopathy. We used in vivo and in vitro approaches. For in vivo studies, experimental diabetes was induced in wild-type (WT) mice and 12/15-Lo (also known as Alox15) knockout mice (12/15-Lo−/−); ER stress was then evaluated after 12-14 weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb) (Nox2−/− mice). In vitro studies were performed using human retinal endothelial cells (HRECs) treated with 15-HETE (0.1 µmol/l) or vehicle, with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress response, NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca2+) release in HRECs treated with or without 15-HETE was performed using confocal microscopy. Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide isomerase (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leukocyte adhesion, VEGFR2 phosphorylation and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of Nox2 had no effect on ER stress induced by the 12/15-LO-derived metabolites both in vitro and in vivo. We also found that 15-HETE increases the intracellular calcium in HRECs. ER stress contributes to 12/15-LO-induced retinal inflammation in diabetic retinopathy via activation of NADPH oxidase and VEGFR2. Perturbation of calcium homeostasis in the retina might also play a role in linking 12/15-LO to retinal ER stress and subsequent microvascular dysfunction in diabetic retinopathy.
  • β-adrenergic receptor/β-arrestin-mediated microRNA maturation regulatory network: A new player in cardioprotective signaling

    Teoh, Jian Peng; Department of Biochemistry and Molecular Biology / Cancer Center (1/25/2018)
    Chronic treatment with the β-blocker carvedilol (Carv) has been shown to reduce established maladaptive left ventricle (LV) hypertrophy and to improve LV function in experimental heart failure. However, the detailed mechanisms by which carvedilol improves LV failure are poorly understood. We previously showed that carvedilol is a β-arrestin-biased β1-adrenergic receptor ligand, which activates cellular pathways through β-arrestins in the heart independent of G protein-mediated second messenger signaling, a concept known as biased signaling. Here, we sought to (i) identify the effects of Carv on LV gene expression on a genome-wide basis and (ii) investigate whether Carv could regulate novel miR expression/biogenesis, thereby providing a novel mechanism for its cardioprotective effects. Gene expression profiling analysis revealed that subsets of genes are differentially expressed after Carv treatment. Further analysis categorized these genes into pathways involved in tight junction, cardiac response to malaria, viral myocarditis, glycosaminoglycan biosynthesis, and arrhythmogenic right ventricular cardiomyopathy (ARVC). Genes encoding proteins in the tight junction, malaria, and viral myocarditis pathways were upregulated in the LV by Carv, while genes encoding proteins in the glycosaminoglycan biosynthesis and ARVC pathways were downregulated by Carv. In addition, our findings also revealed that Carv indeed upregulates 3 mature miRs, but not their pre-miRs and pri-miRs, in a β-arrestin1/2-dependent manner. Interestingly, Carv-mediated activation of miR-466g or miR-532-5p, and miR-674 is dependent on β2AR and β1AR, respectively. Mechanistically, β-arrestins regulate maturation of 3 newly identified βAR/β-arrestin-responsive miRs (β-miRs) by associating with the Dicer complex as well as two RNA binding proteins (hnRNPK and dyskerin) on three pre-miRs. Cardiac cell approaches uncover that β-miRs act as gatekeepers of cardiac cell function by repressing deleterious targets. Our findings indicate a novel role for βAR-mediated β-arrestin signaling activated by Carv in miR maturation, which may be linked to its protective mechanism. Altogether, our findings indicate that (i) the gene expression changes may reflect the molecular mechanisms that underlie the functional benefits of Carv therapy and (ii) the novel role for βAR-mediated β-arrestin signaling activated by Carv in miR maturation, which may be linked to its protective mechanism.

    Sultan, Hussein Mohammed Hussein; Department of Biochemistry and Molecular Biology / Cancer Center (1/25/2018)
    Cytotoxic T lymphocytes (CTLs) are the most effective components of the immune system capable of destroying tumor cells. The identification of CTL tumor epitopes has led to numerous clinical studies in the form of therapeutic vaccines, but the overall immune responses and the antitumor effects of these vaccines have been suboptimal. Previously, The Celis laboratory reported that vaccines prepared with palmitoylated peptides mixed with poly-IC (BiVax) generate strong CTL responses. Poly-IC, a synthetic double stranded RNA, activates both toll like receptor 3 (TLR3) and cytoplasmic melanoma differentiation associated protein 5 (MDA5). Although TLR3 was essential for the priming phase, poly-IC mediated MDA5 activation was critical for the subsequent expansion of the primed T cells upon antigen reencounter (boosting). However, the role of different antigen presenting cells, MDA5 activation and the requirement of type I interferon (IFN-I) and other T cell stimulatory cytokines remained unclear. Here, I describe how dendritic cells (DCs) are essential for palmitoylated peptide presentation and how MDA5 activation regulates the production of IFN-I, which enhances T cell responses indirectly through inducing IL15 and/or IL2 production. Furthermore, blocking IL2 or IL15 signaling inhibited T cell expansion resulting from BiVax immunization. The combination of BiVax with sustained IL2 signaling (provided by administration of IL2/IL2 antibody complexes (BiVax/IL2Cx) or pegylated IL2 generated large endogenous T cell responses that effectively control tumor growth. Furthermore, sustained IL2 signaling improved the inherent ability of antigen specific T cells to resist inhibitory PD-1 signaling. The antitumor effects of BiVax/IL2Cx were associated with severe autoimmune diabetes when using an antigen expressed by the tumor and the pancreas. These symptoms could be avoided while preserving outstanding antitumor responses by utilizing a tumor antigen that is not expressed in the pancreas. Collectively, my study provides a clear evidence that IFN-I and IL2 are important in mediating T cell expansion and enhancing the antitumor effects of peptide-based vaccines. First and foremost, most of the vaccine components have been used in the clinic, and applying this vaccination approach for cancer treatment can be attainable.
  • Deciphering mechanisms of DNA methylation regulation by depletion of the DNA methyltransferases and SETD2

    Tiedemann, Rochelle Lee; Department of Biochemistry and Molecular Biology / Cancer Center (2015)
    DNA methylation (5mC) is a stabile epigenetic mark that confers differential function for gene expression and chromatin accessibility dependent on the context and locality of the mark. Promoter regions populated by CpG islands (CGIs) are highly unmethylated while the remaining ∼80% of CpGs are methylated and distributed across gene bodies, repetitive and transposable elements, and intergenic regions of the genome. The presence and/or absence of particular histone modifications also dictate the patterning of 5mC genome-wide. In cancer, a reversal of 5mC patterns occur in which hypermethylation of tumor suppressor gene CGIs confers gene silencing, and hypomethylation of repetitive and transposable elements contribute to genomic instability. The mechanisms by which 5mC becomes aberrantly regulated in cancer remain unknown. In this study, direct and indirect mechanisms of 5mC regulation were investigated. To understand the direct regulation of 5mC genome-wide, we depleted cell line models of the DNA methyltransferases (DNMTs) that are responsible for establishing (DNMT3A, DNMT3B, DNMT3L) and maintaining (DNMT1) 5mC patterns. Profiling of 5mC patterns on the Illumina HumanMethylation450 BeadChip revealed a unique antithetical relationship between DNMT1 and DNMT3B for the regulation of both 5mC and DNA hydroxymethylation (5hmC) across gene bodies. DNMT3B mediated nonCpG methylation, while DNMT3L influenced the activity of DNMT3B toward nonCG versus CpG site methylation. DNMT3B depletion induced 5mC patterns that closely resemble those observed during cellular differentiation and occurred across gene bodies of highly expressed, H3K36me3-marked genes. SETD2, the histone methyltransferase responsible for H3K36me3 establishment across active gene bodies, was determined to influence the guidance of DNA methylation genome-wide through an indirect mechanism. SETD2 knockout induced widespread loss of H3K36me3 that did not coincide with changes in 5mC. However, paradoxical gains in H3K36me3 significantly induced hypermethylation and upregulation of underlying genes. Genes marked exclusively by the poised enhancer mark, H3K4me1, were commonly targeted for this epigenetic phenotype. DNA methylome profiling of loss-of-function SETD2 mutated clear cell renal cell carcinoma, papillary renal cell carcinoma, and lung adenocarcinoma tumors confirmed the predominance of the hypermethylation phenotype upon loss of SETD2. Collectively, these studies provide novel insight to understanding the regulatory mechanisms by which 5mC patterns are conferred.
  • Role of DNA methyltransferases in maintaining mammary stem/progenitor and cancer stem cells

    Pathania, Rajneesh; Department of Biochemistry and Molecular Biology (2015)
    Breast cancer is the leading cause of cancer death in women worldwide and it affects one in eight women in western countries. Like other human cancers, breast cancer also consists of cellular hierarchy, and heterogeneous. However, the cancer cell of origin and how a normal self-renewal pathway turns into abnormal self-renewal signaling are not known. DNA methylation provides a potential epigenetic mechanism for the cellular memory and heterogeneity, which needed to preserve the tumorigenic potential through repeated cell divisions. Further DNA methylation plays an essential role in stem/progenitor cell maintenance and provides a potential epigenetic mechanism for maintaining cellular memory and heterogeneity during self-renewal. However, the specific role of DNMTs in maintaining mammary stem cells (MaSC) and cancer stem cell (CSC) in a constantly replenishing organ, like mammary glands, is not yet known. Here, we show that Dnmt1 is essential for mammary gland development and indispensable for terminal end bud development and that mammary-gland specific Dnmt1 deletion in mice leads to significant reduction in mammary stem/progenitor cell formation. Moreover, Dnmt1 deletion almost completely abolishes Neu-Tg- and C3(1)-SV40-Tg- driven mammary tumor formation. The reduced tumor incidence observed in Dnmt1 deleted mouse is associated to significant reduction in cancer stem cell formation. These observations were recapitulated using pharmacological inhibitors of DNMTs in Neu-Tg mice in vivo. Further, we show that there is a substantial increase in DNMT1 expression when mammary stem/progenitor cells turn into tumor initiating cells. Using genome-scale methylation approach, we found that hypermethylation of genes involved in development and cell commitment pathways impart immortality and autonomous growth to the cancer stem cells. Moreover, our study provides evidence that stem cells, in addition to luminal progenitor cells, are susceptible for genetic and epigenetic modification and associated with chemotherapeutic resistance. Thus, combination of DNMT and HDAC inhibitors can be used as a therapeutic strategy to block mammary tumor formation and to overcome drug resistance by inhibiting CSCs. These findings improve our understanding of abnormal self-renewal associated with cell of origin, and highlight novel methylation markers that have the potential to serve as useful diagnostic tools and therapeutic targets in early detection of breast cancer.
  • Functions of NF-κB in the Tumor Microenvironment

    Redd, Priscilla Simon; Department of Biochemistry and Molecular Biology (11/7/2017)
    NF-κB is a master transcription factor whose signaling pathway regulates the expression of genes involved in a substantial number of pathways including immunity, cell survival, cell death, inflammation, and proliferation. NF-κB has been shown to promote and suppress tumor development, however, the molecular mechanism underlying this contrasting role of NF-κB is unknown. Our central hypothesis is that the relative functions of NF-κB in immune cells and tumor cells control the dynamic interactions between immune and tumor cells in the tumor microenvironment to dictate tumor progression or regression. To test this hypothesis, we studied NF-κB expression and function in myeloid cells, T cells, and tumor cells. Here we provide evidence for the specific roles of NF-κB in 1) iNOS expression in myeloid and tumor cells and 2) radiation-induced TNFα and Fas expression in tumor cells. In the first publication, we found that iNOS is expressed in colon carcinoma cells and tumor-infiltrating immune cells. We not only show that NF-κB is specifically binding to the nos2 promoter in both human colon carcinoma cells and murine myeloid cells, but specifically a p65/p65 and p50/p50 homodimer is binding to the nos2 promoter in myeloid cells. In the second publication, we found that radiation induces rapid activation of NF-κB in human soft tissue sarcoma. Specifically, radiation is activating the p65/p50 heterodimer and p50/p50 homodimer that is then binding to TNFα and FAS promoter sequences. Using a syngeneic mouse model, irradiated tumors had a significant reduction in tumor growth and induced a Th1/Tc1 T cell response, shown by an increase in signature genes of T cells in which NF-κB has been reported to be a transcriptional activator. NF-κB therefore acts as a molecular link between tumor cells and immune cells in the tumor microenvironment in irradiated tumors. In summary, our data indicate that the different NF-κB dimers regulate the expression of both tumor suppressors such as Fas and tumor promoters such as iNOS. Therefore, the cellular context-dependent NF-κB dimer composition might underlie the contrasting NF-κB functions in tumor promotion or suppression through regulating the expression of genes with opposite functions during tumor development.
  • Polyfunctional CD4+ T cells synergize with chemotherapy to reprogram tumor metabolism towards a curative outcome

    Habtetsion, Tsadik Ghebreamlak; Department of Biochemistry and Molecular Biology (8/3/2017)
    CD4+ T cells are critical mediators of anti-tumor immunity. Accumulating evidence from preclinical and clinical studies suggests that tumor-reactive CD4+ T cells in adoptive T cell therapy (ACT) have the potential to effectively control tumor growth. In most ACT clinical settings, chemotherapeutic agents are used to induce an immunostimulatory milieu which facilitates the effector function of donor T cells. Although the efficacy of ACT has been well-established, currently only a fraction of patients with certain types of malignancy have benefited, highlighting the need for improved ACT strategies. Recent studies have revealed that the metabolic reprogramming by cancer cells attenuate antitumor immune response by imposing nutrient restrictions in the tumor microenvironment, which leads to defective T cell responses. In the current study, we set out to explore how Cyclophosphamide (CTX) and tumor reactive CD4+ T cells alter the metabolic features of cancer cells. By comparing the global metabolic profiling of tumors pre and post-treatment, we found that CXT+CD4 ACT elicited a metabolic catastrophe affecting multiple pathways critical for cancer progression. Particularly, CTX+CD4 ACT led to marked reduction in glutathione (GSH) levels, increased accumulation of reactive oxygen species (ROS) and oxidative DNA damage product in tumors. Importantly, administration of N-acetyl-L-cysteine diminished the curative effect of CTX+CD4 ACT. Moreover, pharmacological inhibition of GSH using Buthionine Sulfoximine (BSO) following CTX significantly delayed tumor growth in mice. Mechanistically, we found that TNFα synergized with chemotherapy to reduce intracellular GSH levels and promote ROS induction. TNFα enhanced cell death in chemotherapy pre-treated tumor cells and the cytotoxic effect was reversed by adding GSH exogenously. Importantly, the curative effect of CTX+CD4 ACT was abrogated after TNFα neutralization. Additionally, we found that CTX+CD4 ACT led to tumor vascular disruption causing hemorrhagic necrosis of tumors. IFNγR-deficient mice failed to reject tumor after CTX+CD4 ACT and had intact tumor vasculature. Collectively, our data reveal that tumor reactive CD4+T cells disrupt the redox homeostasis of cancer cells in TNFα-dependent manner. Whereas CD4+T cells derived IFNγ targeted tumor endothelial cells to cause vascular disruption and tissue ischemia. The combined action of these two cytokines leads to eventual eradication of established tumor.
  • The Differential Roles of PI3K P110 Isoforms in Regulating CD4 T Cell Subset Polarization

    Webb, Mason James; Department of Biochemistry and Molecular Biology (5/12/2017)
    Class IA phosphatidylinositol-4,5-bisphosphate 3-kinases, or PI3K’s, are one of the earliest bottlenecks for T cell receptor signaling transduction, without which phosphorylated phosphatidylinositides cannot be generated and the T cell activation cascade becomes impaired. Of the catalytic class IA PI3K subunits, there are three isoforms designated as p110α, p110β, and p110δ. The Khleif laboratory has discovered that these catalytic subunits display unique roles in T regulatory cells and non-polarized activated CD4+ T cells. This thesis aims to determine what differential control these p110 isoforms have upon distinct polarized CD4+ T cell subsets.
  • The Regulation of y-globin by microRNA

    Ward, Christina Marie Torres; Department of Biochemistry and Molecular Biology (2016)
    High fetal hemoglobin (HbF) attenuates the severity of sickle cell disease (SCD) by interfering with hemoglobin S polymerization; therefore increasing yglobin expression is an effective therapeutic approach for SCD. Variations in HbF levels in sickle cell patients are associated with inherited genetic differences found in genes encoding regulatory factors throughout the genome involved in yglobin gene regulation. Molecular targets such as microRNAs (miRNAs) that modulate gene expression through post-transcriptional mechanisms are under investigation as novel y-globin regulators. The goal of this project is to identify miRNA genes which regulate HbF. First, computational in silico analysis was performed which identified miR-34a as a predicted target in the y-globin gene 3'untranslated region (UTR). Transient and stable expression of miR-34a in K562 cells, demonstrated increased y-globin transcription and HbF synthesis. We observed increased expression ofGATAl, KLFI , CD235a, and the erythropoietin receptor supporting the promotion of erythroid differentiation by miR-34a. The fact that miR-34a activated HbF suggest it targets a negative regulator of y-globin gene expression rather than targeted the 3 'UTR to promote degradation. The levels of total and phosphorylated STA T3 were decreased in the miR-34a stable clones suggesting a role of the y-globin repressor ST A T3, in the mechanism of HbF activation by miR-34a. For the second approach, we analyzed miRNA profiles using reticulocytes isolated from individuals with SCD with low and high HbF levels and identified miR-144 as highly expressed in the low HbF group. miR-144 targets NRF2, a transcription factor that mediates the cellular oxidative stress response and is involved in drug-mediated HbF induction. Primary erythroid progenitors in culture treated with miR-144 mimic had a decrease in the number of HbF positive cells and the levels of NRF2 and HbF protein. Pretreatment with miR-144 inhibitor reversed its negative effect on HbF expression. Further evidence for this novel mechanism of y-globin regulation was collected using KU812 cells treated with hemin to generate a tissue culture model of oxidative stress. The robust activation of NRF2 and HbF induction by hemin was abolished after pretreatment with miR-144 inhibitor. These data support miR- 34a and miR-144, as positive and negative regulators respectively of HbF expression.
  • Combinational immunotherapy of anti-OX40 antibody and IDO inhibitor synergistically enhances anti-tumor immune T cell-mediated response

    Berrong, Zuzana Jirina; Department of Biochemistry and Molecular Biology (2016-05)
    One of the major goals of cancer immunotherapy is to disrupt the immunosuppressive environment that allows tumors to thrive in and to generate potent and enduring antitumor specific-immune responses. Cancer vaccines may elicit antigen-specific immune responses; however, this is, in many tumor models and in human cancer, insufficient for positive outcomes due to existence of multiple immune-inhibitory mechanisms in tumors. A relatively recently introduced strategy to increase the therapeutic efficacy of tumor vaccination is to combine different immunological approaches that target different immunosuppressive pathways and to enhance the efficacy of vaccines by T cell agonists. OX40 is a co-stimulatory receptor expressed on T cells that can lead to proliferation and enhancement ofT cell effector function when bound by its ligand or targeted with agonist antibody. Here, we show that different doses of anti-OX40 antibody (Ab) elicit differential impacts on the T cell immune response resulting in either efficacious or detrimental therapeutic effect in immunized tumor-bearing mice. We demonstrate that treating tumor-bearing mice with an optimal dose of 1 mg/kg anti-OX40 Ab leads to a potent therapeutic and immune anti-tumor effect when combined with vaccine, whereas higher dose at 2.5 mg/kg of anti-OX40 Ab with vaccine increases the accumulation of regulatory T cells in the tumor and diminishes the therapeutic effect. Furthermore, we proposed that OX40 downstream molecular signaling through AKT activation in T cells may elucidate the differential T cell response when stimulated with anti-OX40 Ab. After optimizing the dose of agonist anti-OX40 Ab to stimulate the immune system toward maximal anti-tumor response when combined with vaccine, we strategized to improve the combinational therapy by targeting the so far untouched immunosuppressive environment. One of the immune suppressive molecules correlating with cancer progression is indoleamine-(2,3)-dioxygenase (IDO) enzyme. The catalytic activity of IDO hinders effector T cells from properly eliciting an anti-tumor effect. Here, we further evaluated the therapeutic outcome and immune mechanisms of the vaccineinduced immune response enhancement by agonist anti-OX40 antibody, while inhibiting the immunosuppressive IDO enzyme. We demonstrate that therapeutic efficacy of this combinational treatment leads to a profound inhibition of tumor growth and complete regression of established tumors in 60% of treated mice. We show that the mechanisms responsible for this therapeutic potency are: i) an increase in vaccine-induced tumorinfiltrating effector T cells that is facilitated by anti-OX40 antibody, and ii) a decrease of IDO enzyme activity within the tumor and the enhancement in the functionality of effector T cells that are facilitated by 1-methyl tryptophan (1-MT, IDO inhibitor). Our findings provide a promising and translatable strategy that can enhance the overall efficacy of cancer immunotherapy.
  • Super enhancer-associated molecular signatures reveal a dependency on immune and metabolic mechanisms in chronic lymphatic leukemia

    Shull, Austin Y.; Department of Biochemistry and Molecular Biology (7/12/2016)
    Chronic lymphocytic leukemia (CLL), characterized by the progressive and uncontrolled accumulation of CD19+ B cells, currently remains as an incurable malignancy despite recent advancements in treatment options. The difficulties of eliciting curative measures in CLL are partly driven by the adaptability of the transcriptional response in CLL cells. In this study, we sought to better understand the complexities of the CLL transcriptional profile by defining the large histone H3 lysine-27 acetylation regions known as “super enhancers” within B cells and determining which genes overexpressed in CLL overlapped with super enhancers. From this analysis, we identified 190 super enhancer-associated genes overexpressed in CLL and determined that many of the genes identified were either involved in immune signaling cascades (e.g. LCK, FCER2) or metabolic regulation (e.g. LSR, ENO2). These processes corresponded with our reverse phase protein array (RPPA) profile of CLL patients, which shows overexpression of immune signaling kinases (e.g. LCK) as well as alteration of metabolically sensitive translation regulators (e.g. 4E-BP1 phosphorylation). Additionally, we determined that CLL cells are apoptotically sensitive to dual PI3K/mTOR inhibition when compared to upstream B cell receptor pathway inhibition due to their differential effects on 4E-BP1 phosphorylation. Based on the derived information from our super enhancer expression signature, we then compared the effects of preferentially targeting super enhancers with either the BET bromodomain inhibitor JQ1 or the cyclin dependent kinase-7 (CDK7) inhibitor THZ1. From this comparison, we saw that JQ1 could inhibit cell cycle progression in CLL cell lines as well as differentially disrupt transcription of genes involved in immune signaling. Contrastingly, we saw that THZ1 elicited a different response in CLL cells by inducing apoptosis and differentially downregulating genes involved in metabolism. The specific super enhancer-associated genes disrupted by the respective treatments further highlighted the dichotomy of JQ1 and THZ1-mediated effects, as JQ1 suppressed the B cell activation marker gene FCER2 whereas THZ1 suppressed the glycolytic enolase gene ENO2. Collectively, these results reveal that super enhancers play a role in mediating both immune signaling and metabolic expression signatures in CLL and that super enhancers can be differentially disrupted by BET bromodomain or CDK7 inhibition.
  • Modulation of indoleamine 2, 3-dioxygenase 1 expression by activated Tcells in breast cancer is controlled by epigenetic mechanisms

    Noonepalle, Satish Kumar Reddy; Department of Biochemistry and Molecular Biology (2015-09)
    Tumor infiltrating lymphocytes (TILs) secrete cytokines that modulate immune responses at the tumor microenvironment. Tumor suppressor activity of interferon gamma (IFNy) cytokine also activates expression of immune suppressive factors such as IDO and PD-L1 in tumor cells. However, there is still much to learn about how tumor cells counter the immune cells at the gene expression level. In this study, RNA-seq analysis of breast cancer cells after in-vitro co-culture with anti-CD3/CD28 activated human T -cells revealed that the IFNy induced immune response gene signature is common to both triple negative breast cancer (TNBC) MDA-MB-231 and estrogen receptor positive (ER+) MCF7 cells. However, IDOl expression was differentially upregulated with significantly higher expression in MDA-MB-231 compared to MCF7 cells. Analysis of the TCGA breast invasive carcinoma dataset revealed subtype specific mRNA expression and IDOl promoter DNA methylation. We observed that IDOl mRNA expression and promoter methylation followed inverse correlation. TNBC/Basal subtype was hypomethylated at the IDOl promoter with higher mRNA expression compared to the ER+ subtype that was hypermethylated with relatively lower IDOl mR.NA expression. The IDOl promoter methylation was confirmed by pyrosequencing analysis of a panel of breast cancer cell lines and patient tumors. IFNy treatment of MDA-MB-231 and MCF7 breast cancer cells revealed no difference in terms of upstream signaling and IDOl mRNA stability. Treatment with demethylating agent, 5-azadeoxycytidine, synergistically up-regulated IDOl mRNA expression in ER+ MCF7 cells highlighting that CpG methylation controls !DO 1 gene expression. We also found a positive correlation between !DO I and CDBA expression and better relapse free survival in TNBC/basal subtype patients suggesting that !DO 1 expression is driven by intrinsic immune surveillance of TILs. These findings provide evidence that !DO 1 promoter methylation regulates anti-immune responses by tumor cells towards TILs and it could be used as a predictive biomarker for IDO inhibitor-based immunotherapy of breast cancer.
  • Heat shock protein 70 promotes HCC by modulating DNA-damage response, MAPK/ERK signaling and cellular senescence

    Wang, Yan; Department of Biochemistry and Molecular Biology (2015-10)
    The mechanisms that drive hepatocellular carcinoma (HCC) development are not well understood. Heat shock protein 70 (HSP70) plays a critical role in protein quality control. The HSP70-mediated response has been implicated in the development of different cancer types, however, the detailed mechanisms by which HSP70 supports tumor progression remains to be investigated. In this research work we observed that HSP70 deletion impairs HCC development by modulating the carcinogen-induced DNA damage response. This results in increased sensitivity to p53-dependent apoptosis, activation of MAPK/ERK negative feedback signaling pathway, and induction of cellular senescence. Inactivation of HSP70 may be a strategy to interfere with signaling pathways that drive liver cancer progression thus offering a therapeutic possibility for human HCC treatment. Note: The research data described in this Ph.D. Thesis are not published. Additional experimental work is needed to verify the data and solidify the mechanistic conclusions of this work before we seek publication of the data in a peer reviewed scientific journal. In light of new data generated from additional studies, we may need to modify or revised our mechanistic conclusions.
  • The Role of GCN2-Dependent Metabolic Stress in Type-2 Inflammation Within the Lung

    Bradley, Jillian; Department of Biochemistry and Molecular Biology (2015-06)
    Determining the control mechanisms behind the development of type-1 and type-2 inflammation could hand us the key to treating inflammatory pathologies like asthma, allergy, and cancer. One potential mechanism is the IDO-GCN2 nutrient stress-sensing pathway that has been implicated during pregnancy, bacterial infections, and others. We have determined that during type-2 inflammation IDO is induced and depletes the environment of tryptophan, this lack of tryptophan then activates GCN2. GCN2, especially when activated in the M2 macrophage subset, helps to regulate the cytokine production, cellular infiltrate, antibody production, and T-cell proliferation, activation, and differentiation. These effect culminate into regulating the overall lung pathology. This study suggests a novel type-2 inflammatory control mechanism through the GCN2 signaling pathway, and provides a potential therapeutic target for treatment of type-2 pathologies, like helminth infection, asthma, allergy, and cancers.
  • AKAP350 Targets to the Golgi Apparatus Where it Interacts with CLIC5B and CIP4/S

    Shanks, Ryan; Department of Biochemistry and Molecular Biology (2002-03)
    A-kinase anchoring proteins (AKAPs) are defined by their ability to scaffold PKA, but their function depends upon their targeting of PKA and other scaffolded signaling proteins to specific subcellular compartments. We have investigated one AKAP, AKAP350, which can scaffold a number of protein kinases and phosphatases at the centrosome and the Golgi apparatus. The AKAP350 gene is multiply spliced to create three carboxyl terminal splice variants which we have designated AKAP350A, AKAP350B and AKAP350C. Immunocytochemistry in HCA-7 cells demonstrated that AKAP350A was localized specifically to the Golgi apparatus. GFP-fusion proteins representing the carboxyl terminus of AKAP350A identified a carboxyl terminal region responsible for the Golgi apparatus targeting of AKAP350A. Yeast two-hybrid analysis was utilized to screen a rabbit gastric parietal cell library with a 3.2kb segment of AKAP350 (nucleotides 3611-6813) which is weakly homologous to pericentrin. This screen yielded two positive clones representing rabbit chloride intracellular channel 1 (CLIC1) and rabbit Cdc42 interacting protein 5 (CIP5). Further yeast-two hybrid binary analysis determined that CLIC1 and CIP5 bound to AKAP350 through adjacent domains located with in the PHR. CLIC1 belongs to a family of proteins which all contain a high degree of homology in their carboxyl termini, and this conserved domain is responsible for several CLIC family member’s ability to bind AKAP350. We isolated the human homologue of bovine p64, CLIC5B, from an HCA-7 colonic adenocarcinoma cell cDNA. A splice variant of CLIC5, the predicted molecular weight of CLIC5B corresponds to the molecular weight of a major CLIC immunoreactive protein in HCA-7 cells. Immunocytochemistry determined that CLIC5B colocalized with AKAP350 at the Golgi apparatus. Yeast-two hybrid binary analysis determined that the final 120 amino acids of CLIC5B interacted with AKAP350. Furthermore, expression of a GFP-fusion protein containing the final 120 amino acids targeted to the Golgi apparatus in HCA-7 cells. CIP5 contains high homology to the human protein Cdc42 interacting protein 4 (CIP4). Yeast-two hybrid binary analysis determined that the first 117 amino acids of both human CIP4 and CIP5 interacted with AKAP350. Immunocytochemistry in HCA-7 cells determined with an antibody recognizing CIP4 and CIP5 localized to the Golgi apparatus. These results suggest that AKAP350 associates with CLIC proteins and CIP4/5, and these proteins interact with the AKAP35A splice variant at the Golgi apparatus.
  • AKAP350: A Centrosome Associated Scaffold Protein

    Schmidt, Hank; Department of Biochemistry and Molecular Biology (2000-06)
    A-kinase anchoring proteins (AKAPs) are recognized as key components of compartmentalization and transduction in intracellular cAMP signaling. They allow localization of the Type II c AMP-dependent protein kinase to specific subcellular domains, effectively positioning the enzyme near its substrate to await activation by cAMP. The role of AKAPs as protein scaffolds allows binding of multiple enzymes, regulatory molecules, and structural elements, functioning as a virtual platform for modulation of specific cellular events (i.e. membrane channel activity, receptor clustering). We have cloned a novel 350 kDa AKAP (AKAP350) from human gastric cDNA, and identified partial clones in human lung and rabbit parietal cells. The genomic region containing AKAP350, found on chromosome 7q21, is multiply spliced, producing at least three distinct AKAP350 isoforms as well as yotiao, an NMDA receptorassociated protein. We identified three unique AKAP350 C-termini (AKAP350A, -B, and -C) resulting from alternative splicing of the 3' end of the gene. AKAP350 is associated with centrosomes, as well as with the cleavage furrow during anaphase and telophase by immunocytochemistry. Polyclonal antibodies to individual AKAP350 Cterminal splice variants demonstrate tissue dependent combinations of centrosomal and non-centrosomal distribution. In the polarized HCA-7 colon cell line AKAP350A is purely non-centrosomal while AKAP350B and -C are centrosomal. Anti-AKAP350C is limited to mitotic cells, suggesting that this isoform may be expressed only at entry into M phase. A yeast two-hybrid screen of a rabbit parietal cell library identified a novel TACC (Transforming Acidic Coiled coil Containing) protein family member as a ligand of the final pair of arginine residues in the AKAP350A splice variant. A GFP fusion with the novel AKAP interacting protein verified co-localization with AKAP350 at the centrosome exclusively during mitosis. Microinjection of dividing sea urchin embryos with GST fused to the AKAP interacting protein arrested cell division. Therefore, the AKAP350 protein scaffold may function as a large docking station, providing kinase / phosphatase signals for coordination of cytoskeletal dynamics as well as cell division.

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