Matching Items (113)
Description
Almost every form of cancer deregulates the expression and activity of anabolic glycosyltransferase (GT) enzymes, which incorporate particular monosaccharides in a donor acceptor as well as linkage- and anomer-specific manner to assemble complex and diverse glycans that significantly affect numerous cellular events, including tumorigenesis and metastasis. Because glycosylation is not template-driven, GT deregulation yields heterogeneous arrays of aberrant intact glycan products, some in undetectable quantities in clinical bio-fluids (e.g., blood plasma). Numerous glycan features (e.g., 6 sialylation, β-1,6-branching, and core fucosylation) stem from approximately 25 glycan “nodes:” unique linkage specific monosaccharides at particular glycan branch points that collectively confer distinguishing features upon glycan products. For each node, changes in normalized abundance (Figure 1) may serve as nearly 1:1 surrogate measure of activity for culpable GTs and may correlate with particular stages of carcinogenesis. Complementary to traditional top down glycomics, the novel bottom-up technique applied herein condenses each glycan node and feature into a single analytical signal, quantified by two GC-MS instruments: GCT (time-of-flight analyzer) and GCMSD (transmission quadrupole analyzers). Bottom-up analysis of stage 3 and 4 breast cancer cases revealed better overall precision for GCMSD yet comparable clinical performance of both GC MS instruments and identified two downregulated glycan nodes as excellent breast cancer biomarker candidates: t-Gal and 4,6-GlcNAc (ROC AUC ≈ 0.80, p < 0.05). Resulting from the activity of multiple GTs, t-Gal had the highest ROC AUC (0.88) and lowest ROC p‑value (0.001) among all analyzed nodes. Representing core-fucosylation, glycan node 4,6-GlcNAc is a nearly 1:1 molecular surrogate for the activity of α-(1,6)-fucosyltransferase—a potential target for cancer therapy. To validate these results, future projects can analyze larger sample sets, find correlations between breast cancer stage and changes in t-Gal and 4,6-GlcNAc levels, gauge the specificity of these nodes for breast cancer and their potential role in other cancer types, and develop clinical tests for reliable breast cancer diagnosis and treatment monitoring based on t-Gal and 4,6-GlcNAc.
ContributorsZaare, Sahba (Author) / Borges, Chad (Thesis director) / LaBaer, Joshua (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Disturbances in the protein interactome often play a large role in cancer progression. Investigation of protein-protein interactions (PPI) can increase our understanding of cancer pathways and will disclose unknown targets involved in cancer disease biology. Although numerous methods are available to study protein interactions, most platforms suffer from drawbacks including high false positive rates, low throughput, and lack of quantification. Moreover, most methods are not compatible for use in a clinical setting. To address these limitations, we have developed a multiplexed, in-solution protein microarray (MISPA) platform with broad applications in proteomics. MISPA can be used to quantitatively profile PPIs and as a robust technology for early detection of cancers. This method utilizes unique DNA barcoding of individual proteins coupled with next generation sequencing to quantitatively assess interactions via barcode enrichment. We have tested the feasibility of this technology in the detection of patient immune responses to oropharyngeal carcinomas and in the discovery of novel PPIs in the B-cell receptor (BCR) pathway. To achieve this goal, 96 human papillomavirus (HPV) antigen genes were cloned into pJFT7-cHalo (99% success) and pJFT7-n3xFlag-Halo (100% success) expression vectors. These libraries were expressed via a cell-free in vitro transcription-translation system with 93% and 96% success, respectively. A small-scale study of patient serum interactions with barcoded HPV16 antigens was performed and a HPV proteome-wide study will follow using additional patient samples. In addition, 15 query proteins were cloned into pJFT7_nGST expression vectors, expressed, and purified with 93% success to probe a library of 100 BCR pathway proteins and detect novel PPIs.
ContributorsRinaldi, Capria Lakshmi (Author) / LaBaer, Joshua (Thesis director) / Mangone, Marco (Committee member) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
The TP53 tumor suppressor gene is the most frequently mutated gene in human cancers. In the highly aggressive triple negative breast cancer (TNBC), TP53 is mutated in 80% of cases. TNBC lacks viable drug targets, resulting in a low prognosis (12.2% 5 year survivability rate). As such, the discovery of druggable targets in TNBC would be beneficial. Mutated p53 protein typically occurs as a missense mutation and often endows cancer cells with gain of function (GOF) properties by dysregulating metabolic pathways. One of these frequently dysregulated pathways is the Hippo/Yes-associated protein-1 (YAP1)/WW Domain Containing Transcription Regulator 1 (TAZ) tumor suppressor pathway. This study therefore analyzed the involvement of the Hippo/YAP1/TAZ pathway in p53-mediated breast cancer cell invasion. From an RNA-seq screen in MCF10A cell lines harboring different TP53 missense mutations, each with a differing invasive phenotype, components of the Hippo pathway were found to correlate with cell invasion. To this end, the active and inactive forms of YAP1 and TAZ were studied. Phosphorylated (inactive) YAP1 and TAZ are retained in the cytoplasm and eventually degraded. Unphosphorylated (active) YAP1 and TAZ translocate to the nucleus to activate TEAD-family transcription factors, inducing cell survival and proliferation genes leading to increased cell invasion. Using quantitative western blot analysis, it was found that inactive TAZ expression was lower in the most invasive cell lines and higher in the least invasive cell lines (p = 0.003). Moreover, the ratio of inactive TAZ protein to total TAZ protein was also shown to be predominantly lower in the invasive cell lines compared to the non-invasive lines (p = 0.04). Finally, active TAZ expression was primarily higher in p53-mutant invasive cell lines and lower in non-invasive p53 mutant cells. Additionally, although YAP1 and TAZ are thought to be functionally redundant, the pattern seen in TAZ was not seen in the YAP1 protein. Taken together, the results demonstrated here suggest that TAZ holds a more dominant role in governing TNBC cell invasion compared to YAP1 and further highlights TAZ as a potential therapeutic target in TNBC.
ContributorsGrief, Dustin (Author) / LaBaer, Joshua (Thesis advisor) / Anderson, Karen (Committee member) / Nikkhah, Mehdi (Committee member) / Arizona State University (Publisher)
Created2022
Description
Glioblastoma (GBM), the most common and aggressive primary brain tumor affecting adults, is characterized by an aberrant yet druggable epigenetic landscape. The Histone Deacetylases (HDACs), a major family of epigenetic regulators, favor transcriptional repression by mediating chromatin compaction and are frequently overexpressed in human cancers, including GBM. Hence, over the last decade there has been considerable interest in using HDAC inhibitors (HDACi) for the treatment of malignant primary brain tumors. However, to date most HDACi tested in clinical trials have failed to provide significant therapeutic benefit to patients with GBM. This is because current HDACi have poor or unknown pharmacokinetic profiles, lack selectivity towards the different HDAC isoforms, and have narrow therapeutic windows. Isoform selectivity for HDACi is important given that broad inhibition of all HDACs results in widespread toxicity across different organs. Moreover, the functional roles of individual HDAC isoforms in GBM are still not well understood. Here, I demonstrate that HDAC1 expression increases with brain tumor grade and is correlated with decreased survival in GBM. I find that HDAC1 is the essential HDAC isoform in glioma stem cells and its loss is not compensated for by its paralogue HDAC2 or other members of the HDAC family. Loss of HDAC1 alone has profound effects on the glioma stem cell phenotype in a p53-dependent manner and leads to significant suppression of tumor growth in vivo. While no HDAC isoform-selective inhibitors are currently available, the second-generation HDACi quisinostat harbors high specificity for HDAC1. I show that quisinostat exhibits potent growth inhibition in multiple patient-derived glioma stem cells. Using a pharmacokinetics- and pharmacodynamics-driven approach, I demonstrate that quisinostat is a brain-penetrant molecule that reduces tumor burden in flank and orthotopic models of GBM and significantly extends survival both alone and in combination with radiotherapy. The work presented in this thesis thereby unveils the non-redundant functions of HDAC1 in therapy- resistant glioma stem cells and identifies a brain-penetrant HDACi with higher selectivity towards HDAC1 as a potent radiosensitizer in preclinical models of GBM. Together, these results provide a rationale for developing quisinostat as a potential adjuvant therapy for the treatment of GBM.
ContributorsLo Cascio, Costanza (Author) / LaBaer, Joshua (Thesis advisor) / Mehta, Shwetal (Committee member) / Mirzadeh, Zaman (Committee member) / Mangone, Marco (Committee member) / Paek, Andrew (Committee member) / Arizona State University (Publisher)
Created2022
Description
Transient Receptor Potential Vanilloid-1 (TRPV1) is an integral membrane polymodal cation channel involved in various essential biological functions, including thermosensing, thermoregulation, and nociception. Discrete TRPV1 activation modes such as ligand, heat, and proton have been challenging to disentangle. However, dissecting the polymodal nature of TRPV1 is essential for therapeutic development. The human TRPV1 (hTRPV1) voltage-sensing like domain (VSLD; transmembrane helices S1-S4) contains the canonical vanilloid ligand binding site and significantly contributes to thermosensing. Nuclear magnetic resonance (NMR)-detected studies probe the role of the hTRPV1-VSLD in TRPV1 polymodal function. The hTRPV1-VSLD is identified as an allosteric hub for all three primary TRPV1 activation modes and demonstrates plasticity in chemical ligand modulation. The presented results underscore molecular features in the VSLD that dictate TRPV1 function, highlighting important considerations for future therapeutic design.
ContributorsOwens, Aerial M. (Author) / Van Horn, Wade D. (Thesis advisor) / Levitus, Marcia (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2023
Description
The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic, declared in March 2020 resulted in an unprecedented scientific effort that led to the deployment in less than a year of several vaccines to prevent severe disease, hospitalizations, and death from coronavirus disease 2019 (COVID-19). Most vaccine models focus on the production of neutralizing antibodies against the spike (S) to prevent infection. As the virus evolves, new variants emerge that evade neutralizing antibodies produced by natural infection and vaccination, while memory T cell responses are long-lasting and resilient to most of the changes found in variants of concern (VOC). Several lines of evidence support the study of T cell-mediated immunity in SARS-CoV-2 infections. First, T cell reactivity against SARS-CoV-2 is found in both (cluster of differentiation) CD4+ and CD8+ T cell compartments in asymptomatic, mild, and severe recovered COVID-19 patients. Second, an early and stronger CD8+ T cell response correlates with less severe COVID-19 disease [1-4]. Third, both CD4+ and CD8+ T cells that are reactive to SARS-CoV-2 viral antigens are found in healthy unexposed individuals suggesting that cross-reactive and conserved epitopes may be protective against infection.
The current study is focused on the T cell-mediated response, with special attention to conserved, non-spike-cross-reactive epitopes that may be protective against SARS-CoV-2. The first chapter reviews the importance of epitope prediction in understanding the T cell-mediated responses to a pathogen. The second chapter centers on the validation of SARS-CoV-2 CD8+ T cell predicted peptides to find conserved, immunodominant, and immunoprevalent epitopes that can be incorporated into the next generation of vaccines against severe COVID-19 disease. The third chapter explores pre-existing immunity to SARS-CoV-2 in a pre-pandemic cohort and finds two highly immunogenic epitopes that are conserved among human common cold coronaviruses (HCoVs). To end, the fourth chapter explores the concept of T cell receptor (TCR) cross-reactivity by isolating SARS-CoV-2-reactive TCRs to elucidate the mechanisms of cross-reactivity to SARS-CoV-2 and other human coronaviruses (HCoVs).
ContributorsCarmona, Jacqueline (Author) / Anderson, Karen S (Thesis advisor) / Lake, Douglas (Thesis advisor) / Maley, Carlo (Committee member) / Mangone, Marco (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2023
Description
Redox homeostasis is described as the net physiologic balance between inter-convertible oxidized and reduced equivalents within subcellular compartments that remain in a dynamic equilibrium. This equilibrium is impacted by reactive oxygen species (ROS), which are natural by-products of normal cellular activity. Studies have shown that cancer cells have high ROS levels and altered redox homeostasis due to increased basal metabolic activity, mitochondrial dysfunction, peroxisome activity, as well as the enhanced activity of NADPH oxidase, cyclooxygenases, and lipoxygenases. Glioblastoma (GBM) is the most prevalent primary brain tumor in adults with a median survival of 15 months. GBM is characterized by its extreme resistance to therapeutic interventions as well as an elevated metabolic rate that results in the exacerbated production of ROS. Therefore, many agents with either antioxidant or pro-oxidant mechanisms of action have been rigorously employed in preclinical as well as clinical settings for treating GBM by inducing oxidative stress within the tumor. Among those agents are well-known antioxidant vitamin C and small molecular weight SOD mimic BMX-001, both of which are presently in clinical trials on GBM patients. Despite the wealth of investigations, limited data is available on the response of normal brain vs glioblastoma tissue to these therapeutic interventions. Currently, a sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was established for the quantification of a panel of oxidative stress biomarkers: glutathione (GSH), cysteine (Cys), glutathione disulfide (GSSG), and cysteine disulfide in human-derived brain tumor and mouse brain samples; this method will be enriched with additional oxidative stress biomarkers homocysteine (Hcy), methionine (Met), and cystathionine (Cyst). Using this enriched method, we propose to evaluate the thiol homeostasis and the redox state of both normal brain and GBM in mice after exposure with redox-active therapeutics. Our results showed that, compared to normal brain (in intact mice), GBM tissue has significantly lower GSH/GSSG and Cys/CySS ratios indicating much higher oxidative stress levels. Contralateral “normal” brain tissue collected from the mice with intracranial GBM were also under significant oxidative stress compared to normal brains collected from the intact mice. Importantly, normal brain tissue in both studies retained the ability to restore redox homeostasis after treatment with a redox-active therapeutic within 24 hours while glioblastoma tissue does not. Ultimately, elucidating the differential redox response of normal vs tumor tissue will allow for the development of more redox-active agents with therapeutic benefit.
ContributorsShaik, Kamal (Author) / LaBaer, Joshua (Thesis director) / Tovmasyan, Artak (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Historical, Philosophical & Religious Studies, Sch (Contributor) / Dean, W.P. Carey School of Business (Contributor)
Created2022-12
Description
DNA methylation (DNAm) is an epigenetic mark with a critical role in regulating gene expression. Altered clinical states, including toxin exposure and viral infections, can cause aberrant DNA methylation in cells, which may persist during cell division. Current methods to study genome-wide methylome profiles of the cells require a long processing time and are expensive. Here, a novel technique called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), which is amenable to automation. Up to 15 different samples can be combined into the same run of Mx-MeDIP-Seq, using only 25 ng of DNA per sample. Mx-MeDIP-Seq was used to study DNAm profiles of peripheral blood mononuclear cells (PBMCs) in two biologically distinct RNA viral infections with different modes of transmission, symptoms, and interaction with the host immune system: human immunodeficiency virus1 (HIV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Analysis of 90 hospitalized patients with SARS-CoV-2 and 57 healthy controls revealed that SARS-CoV-2 infection led to alterations in 920 methylated regions in PBMCs, resulting in a change in transcription that affects host immune response and cell survival. Analysis of publicly available RNA-Sequencing data in COVID-19 correlated with DNAm in several key pathways. These findings provide a mechanistic view toward further understanding of viral infections. Genome-wide DNAm changes post HIV-1-infection from 37 chronically ill patients compared to 17 controls revealed dysregulation of the actin cytoskeleton, which could contribute to the establishment of latency in HIV-1 infections. Longitudinal DNAm analysis identified several potentially protective and harmful genes that could contribute to disease suppression or progression.
ContributorsRidha, Inam (Author) / LaBaer, Joshua (Thesis advisor) / Murugan, Vel (Thesis advisor) / Plaisier, Christopher (Committee member) / Nikkhah, Mehdi (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2022
Description
Type 1 diabetes (T1D) is the result of an autoimmune attack against the insulin-producing β-cells of the pancreas causing hyperglycemia and requiring the individual to rely on life-long exogenous insulin. With the age of onset typically occurring in childhood, there is increased physical and emotional stress to the child as well as caregivers to maintain appropriate glucose levels. The majority of T1D patients have antibodies to one or more antigens: insulin, IA-2, GAD65, and ZnT8. Although antibodies are detectable years before symptoms occur, the initiating factors and mechanisms of progression towards β-cell destruction are still not known. The search for new autoantibodies to elucidate the autoimmune process in diabetes has been slow, with proteome level screenings on native proteins only finding a few minor antigens. Post-translational modifications (PTM)—chemical changes that occur to the protein after translation is complete—are an unexplored way a self-protein could become immunogenic. This dissertation presents the first large sale screening of autoantibodies in T1D to nitrated proteins. The Contra Capture Protein Array (CCPA) allowed for fresh expression of hundreds of proteins that were captured on a secondary slide by tag-specific ligand and subsequent modification with peroxynitrite. The IgG and IgM humoral response of 48 newly diagnosed T1D subjects and 48 age-matched controls were screened against 1632 proteins highly or specifically expressed in pancreatic cells. Top targets at 95% specificity were confirmed with the same serum samples using rapid antigenic protein in situ display enzyme-linked immunosorbent assay (RAPID ELISA) a modified sandwich ELISA employing the same cell-free expression as the CCPA. For validation, 8 IgG and 5 IgM targets were evaluated with an independent serum sample set of 94 T1D subjects and 94 controls. The two best candidates at 90% specificity were estrogen receptor 1 (ESR1) and phosphatidylinositol 4-kinase type 2 beta (PI4K2B) which had sensitivities of 22% (p=.014) and 25% (p=.045), respectively. Receiver operating characteristic (ROC) analyses found an area under curve (AUC) of 0.6 for ESR1 and 0.58 for PI4K2B. These studies demonstrate the ability and value for high-throughput autoantibody screening to modified antigens and the frequency of Type 1 diabetes.
ContributorsHesterman, Jennifer (Author) / LaBaer, Joshua (Thesis advisor) / Borges, Chad (Committee member) / Sweazea, Karen (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2022
Description
Antibodies are the immunoglobulins which are secreted by the B cells after a microbial invasion. They are stable and stays in the serum for a long time which makes them an excellent biomarker for disease diagnosis. Inflammatory bowel disease is a type of autoimmune disease where the immune system mistakenly attacks the commensal bacteria and leads to inflammation. We studied antibody response of 100 Crohn’s disease (CD), 100 ulcerative colitis (UC) and 100 healthy controls against 1,173 bacterial and 397 viral proteins. We found some anti-bacterial antibodies higher in CD compared to controls while some antibodies lower in UC compared to controls. We were able to build biomarker panels with AUCs of 0.81, 0.87, and 0.82 distinguishing CD vs. control, UC vs. control, and CD vs. UC, respectively. Subgroup analysis based on the Montreal classification revealed that penetrating CD behavior (B3), colonic CD location (L2), and extensive UC (E3) exhibited highest antibody reactivity among all patients. We also wanted to study the reason for the presence of autoantibodies in the sera of healthy individuals. A meta-analysis of 9 independent biomarker study was performed to find 77 common autoantibodies shared by healthy individuals. There was no gender bias; however, the number of autoantibodies increased with age, plateauing around adolescence. Molecular mimicry likely contributed to the elicitation of a subset of these common autoantibodies as 21 common autoantigens had 7 or more ungapped amino acid matches with viral proteins. Intrinsic properties of protein like hydrophilicity, basicity, aromaticity, and flexibility were enriched for common autoantigens. Subcellular localization and tissue expression analysis indicated the sequestration of some autoantigens from circulating autoantibodies can explain the absence of autoimmunity in these healthy individuals.
ContributorsShome, Mahasish (Author) / LaBaer, Joshua (Thesis advisor) / Borges, Chad (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2021