Recent Submissions


    Naughton, Sean X; Biomedical Sciences (Augusta University, 2019-12)
    Organophosphates (OPs) are a broad class of chemicals with a variety of uses that include pesticides, chemical warfare agents, fuel additives, and plasticizers. Due to their sheer number of applications and known toxicological profile, OPs represent a persistent concern to human health worldwide. Furthermore, the effects of OPs that occur independently of their well-known mechanism of acute toxicity (AChE inhibition) have not been well studied. The presented research seeks to expand upon our understanding of AChE-independent mechanisms of OP toxicity as well as to identify potential therapies for treating these negative effects. In Manuscript 1 we demonstrate that the OP diisopropylfluorophosphate (DFP) induced axonal transport deficits occur in vivo at exposure levels that were not associated with cholinergic toxicity. Additionally, we observed deficits in white matter integrity following sub-acute DFP exposure. In Manuscript 2 we present a series of experiments, which were conducted to identify potential therapeutic compounds for the treatment of OP induced deficits in axonal transport. Here, we utilized a phenotypic drug-screening assay in order to identify compounds that could be protective against DFP. In Manuscript 3 we present data which demonstrates that the carbamate physostigmine does not impair axonal transport, as has been previously demonstrated with OPs. These experiments were critical to demonstrating the AChE independence of OP-induced axonal transport deficits and further elucidate the unique nature of OP toxicity in comparison to other AChE inhibitors. Collectively, these studies contribute to a better understanding of the full spectrum of toxicological effects of OPs and provide insightful findings into potential therapeutics for the treatment of OP related toxicity.
  • Primary Tumor-Induced Immunity Is Suppressed By Surgery-Induced Inflammation In The Presence Of Residual Tumor Cells

    Piranlioglu, Raziye; Biomedical Sciences (Augusta University, 2019-12)
    It is widely thought that tumor cells disseminate from a primary site into the circulation during the early stages of tumor development. However, the fate of these early disseminated tumor cells (DTCs) has been elusive. By utilizing the murine mammary tumors, 4T1 and EMT6, in a syngeneic mouse model, we show that both tumors disseminate into secondary organs but only 4T1 tumors are able to generate metastasis. In contrast, EMT6 primary tumors induce an anti-tumor response that leads to elimination of DTCs. This anti-tumor immunity is CD8+ T cell-dependent and provides long-term immunity. Furthermore, the mice are free of DTCs within a couple of days when primary tumors are completely resected and they reject subsequently injected tumors, whereas mice with residual tumors following surgery show enhanced local recurrence and outgrowth of DTCs at metastatic sites; this effect may be explained by elevated levels of granulocyte colony-stimulating factor (G-CSF). This increase is accompanied by an accumulation of immature myeloid-derived suppressor cells (MDSCs) in the spleen and lungs, the main target organ for metastasis. Moreover, the infiltration of a granulocytic subset of MDSCs (gMDSCs) leads to a decrease in a subset of T cells that have a role in long-term immunity. Our goal for this study is to elucidate how immune components of distant organs affect the fate of DTCs and the role of surgery induced-inflammation in generating a pre-metastatic niche. Our studies may also provide a molecular explanation of improved overall survival in breast cancer patients following complete resection of primary tumors with negative margins.

    Atawia, Reem T.; Biomedical Sciences (Augusta University, 2019-11-05)
    An obesity epidemic continues to rise worldwide. Visceral (central) obesity is an important concern as it correlates with metabolic and cardiovascular pathologies. Arginase is a ureahydrolase enzyme with two isoforms (A1-cytosolic and A2-mitochondrial). We found that visceral adipose tissue (VAT) from obese WT mice fed a high fat/high sucrose diet (HFHS) showed a significantly higher expression of A2 compared to mice fed normal chow diet (ND). We also observed that A2 expression is upregulated 3-fold in differentiated 3T3- L1 adipocytes exposed to high levels of palmitate and glucose, a mimic of the obese state, compared to control media. Our study focused on the involvement of A2 in obesity associated metabolic and vascular disorders. WT mice and those globally lacking A2 (A2-/-) were fed HFHS or ND for 16 weeks. The HFHS diet-induced increases in body and VAT weights and total adiposity were prevented or reduced in A2-/- mice. In concert, metabolic chamber studies revealed that energy expenditure and fatty acid oxidation rates were significantly higher in A2-/- compared to WT HFHS mice. VAT from A2-/- mice fed HFHS had higher levels of active AMPK-α, the master regulator of fatty acid metabolism, as well as higher adipocyte expression of genes involved in fatty acid β-oxidation and oxidative phosphorylation, along with preserved mitochondrial density compared to WT HFHS. A2 deletion also prevented HFHS-induced fibrous tissue deposition and inflammation in VAT, which contributed to adipocyte metabolic dysfunction. These results indicate that A2 is involved in metabolic dysfunctions. To gain insights into the role of A2 in adipocytes, primary preadipocytes isolated from VAT of A2-/- mice and differentiated in vitro showed increased expression of adiponectin and better mitochondrial function. Adenoviral overexpression of A2 in differentiated 3T3-L1 cells showed impaired mitochondrial function and increased mitochondrial ROS. Obesity-related metabolic disorders increase the risk of cardiovascular diseases, the leading global cause of death. Endothelium-dependent vasorelaxation, impaired by HFHS diet, was significantly preserved in A2-/- mice, but more prominently prevented in A1+/- mice. In conclusion, A2 is critically involved in HFHS-induced obesity, VAT inflammation and metabolic dysregulation. Both A1 and A2 are involved in HFHS-induced vascular endothelial dysfunction.

    Ibrahim, Mohammed Mahmoud Labib; Biomedical Sciences (Augusta University, 2019-05)
    A causal link between chronic inflammation and tumorigenesis is now well established in the literature with a great deal of supporting evidences from genetic, epigenetic, pharmacological and epidemiological perspectives. In particular, inflammatory bowel diseases represent an important risk factor for colon cancer development. Moreover, it seems that even sporadic colon cancers that do not develop as a complication of chronic colitis are also driven by inflammation. However, the molecular mechanisms behind inflammation-mediated colon tumorigenesis have remained largely unknown. Colitis associated cancer development is thought to be multifaceted due to a combination of genetic and epigenetic aberrations. Recently, epigenetic alterations -particularly aberrant DNA methylation- have gained great attention in cancer biology and have been observed to play a key role in the pathogenesis of inflammation-associated tumors; especially in colitis-associated cancer. IRF8, a key transcription factor originally identified in myeloid cells, has been reported to play a crucial role in myeloid cells differentiation and immune response regulation. IRF8 deficiency is associated with deregulation of myeloid cell differentiation and accumulation of immature myeloid subsets phenotypically and functionally resemble MDSCs (Myeloid Derived Suppressor Cells). IRF8 is thought to function as a tumor suppressor and was found to be silenced in different types of cancers including colon cancer. Myeloid derived-IRF8 has been extensively studied. However, the role of epithelial-derived IRF8 in colon inflammation and colon cancer initiation remains a point to be addressed. In this study, we generated conditional Irf8cKO mice in which IRF8 is specifically deleted in colon epithelium. Irf8cKO mice exhibit a more aggressive pattern of colitis associated cancer with higher tumor incidence and severe loss of body weight. Additionally, we provide evidence that chronic inflammation promotes the accumulation and infiltration of CD11b+Gr1+ MDSCs, which plentifully secrete IL10 in colon tissue. IL10 then induces STAT3 phosphorylation and nuclear translocation to bind to Dnmt1 and Dnmt3b promoters to upregulate their expression, leading to DNA hyper-methylation at the Irf8 promoter to silence IRF8 expression in colonic epithelial cells and promote colon tumorigenesis. Collectively, our data pinpoint the MDSC-IL10-STAT3-DNMT3b-IRF8 axis as a novel bridge between chronic inflammation and colon cancer formation.
  • The c-MYC oncogene deregulates global DNA methylation and hydroxymethylation to control genome-wide gene expression for tumor maintenance in leukemia/lymphoma

    Poole, Candace Jean; Biomedical Sciences (Augusta University, 2019-05)
    Aberrant DNA methylation is a characteristic feature of tumor cells. However, our knowledge of how DNA methylation patterns are established and maintained to contribute to tumorigenesis is limited. Inactivation of the c-MYC oncogene triggers tumor regression in T-cell acute lymphoblastic leukemia (T-ALL) resulting in dramatic changes to the chromatin landscape including DNA methylation. In this study, I investigated how MYC regulates DNA methylation and hydroxymethylation patterns to contribute to gene expression programs important for tumor maintenance in T-ALL and Burkitt lymphoma. I report that MYC maintains 5-methylcytosine (5mC) and 5-hydroxy-methylcytosine (5hmC) patterns by regulating the DNA methylation machinery, which is important for gene expression in T-ALL. DNA methyltransferases (DNMTs) initiate 5mC marks, while Ten-eleven translocation methylcytosine dioxygenases (TETs) oxidize 5mC to produce 5hmC as an intermediate modification, ultimately leading to active DNA de-methylation. I demonstrated that DNMT1 and DNMT3B are MYC target genes and that their expression is dependent on high MYC levels. Knockdown of DNMT3B in T-ALL reduced cell proliferation through cell cycle arrest and caused the reactivation of gene transcription through reversing promoter/CpG island methylation. Furthermore, I demonstrated that TET1 and TET2 expression is MYC-dependent, as high TET1 and low TET2 levels depend on oncogenic MYC. Knockdown of TET1 in T-ALL reduced cell proliferation through cell cycle arrest and caused genome-wide changes in 5mC and 5hmC corresponding to changes in gene programs important for ribosomal biosynthesis and protein synthesis. In contrast, ectopic expression of TET2 reduced tumor cell proliferation through apoptosis/necrosis and caused genome-wide changes in 5mC and 5hmC corresponding to changes in transcriptional regulatory gene programs. My finding that a coordinated interplay between components of the DNA methylation machinery is necessary for MYC-driven tumor maintenance highlights the potential of targeting specific DNMT or TET proteins for therapeutic strategies.

    Sura, Survasha; Department of Biological Sciences; Department of Biochemical and Molecular Biology; Georgia Cancer Center; Rajpurohit, Surendra K; Augusta University (2019-02-13)
    Zebrafish have emerged as a powerful model organism for elucidating the development and function of microglia. Generation of new transgenic reporter lines and imaging tools strengthen the zebrafish model in microglia study�in-vivo. The aim is to develop a novel compound transgenic line to study the inflammatory process mediated by NF-kB in microglia cells. This novel compound transgenic line will establish a new model for microglia study. To generate the novel compound zebrafish transgenic model for microglia, we are crossbreeding microglia transgenic line zebrafish (Tg(mpeg1:mCherry) with the NF-kB Tg(6xNFkB:EGFP) transgenic progeny. We first generate a heterozygous F1 progeny which will be bred to generate an F2 homozygous progeny. Once the F1 progeny of the Microglia-NfkB transgenic line is developed, they will be crossbred to develop the Homozygous compound transgenic line. Fluorescent Microscopy will be used to screen the larvae generated from the breeding events. By developing the compound transgenic line, we are optimizing microglia isolation and sorting methodology by using the related antibodies as the marker. The NF-kB microglia transgenic line will provide a unique platform for drug screening to address microglial based ailments, thus furthering the understanding and treatment of human disease.
  • The Role of GPR109A in NAD+ Metabolism in Aging RPE

    Fuller, Jasmine; Department of Biochemistry and Cancer Biology (Augusta University, 2018-12-28)
    Age-related macular degeneration (AMD) is the leading cause of blindness in people over the age of 50 worldwide. The retinal pigment epithelium (RPE), located in the back of the eye, is most affected in AMD. Nicotinamide adenine dinucleotide (NAD+) is a coenzyme common to most metabolic pathways. Reductions in NAD+ and NAD+- dependent enzymes (e.g., SIRT1) have been linked causally to the development/progression of many age-related pathologies. None, however, have evaluated NAD+ directly or the mechanisms governing its biosynthesis and related availability in RPE. In our previous study, we have shown that NAD+ levels decline with age in the RPE. This correlated directly with decreased nicotinamide phosphoribosyltransferase (NAMPT) expression. SIRT1 expression and activity was also significantly reduced. Using the human RPE cell line, ARPE-19, primary mouse RPE cells and FK866, a highly specific, noncompetitive NAMPT inhibitor, we simulated in vitro the age-dependent decline in NAD+ and the related increase in RPE senescence. Using this model, we demonstrated the positive impact that therapies that provide supplemental or alternate energy sources such as nicotinamide mononucleotide (NMN) and β-hydroxybutyrate (B-HB) have on RPE viability and the possible role of G-protein coupled receptor, GPR109A in this process.
  • Immune regulation of tumor cell plasticity: A promising molecular target in breast cancer metastasis

    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.

    Angara, Kartik Prasad; Department of Biochemistry and Molecular Biology (Augusta University, 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 (2017)
    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 (2017)
    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.

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