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- All Subjects: Cellular Biology
- Genre: Academic theses
- Creators: Lake, Douglas
- Creators: Kusumi, Kenro
- Member of: ASU Electronic Theses and Dissertations
- Resource Type: Text
Description
Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) is a rare and highly aggressive ovarian cancer that affects children and young women at a mean age of 24 years. Most SCCOHT patients are diagnosed at an advanced stage and do not respond to chemotherapy. As a result, more than 75% of patients succumb to their disease within 1-2 years. To provide insights into the biological, diagnostic, and therapeutic vulnerabilities of this deadly cancer, a comprehensive characterization of 22 SCCOHT cases and 2 SCCOHT cell lines using microarray and next-generation sequencing technologies was performed. Following histological examination, tumor DNA and RNA were extracted and used for array comparative genomic hybridization and gene expression microarray analyses. In agreement with previous reports, SCCOHT presented consistently diploid profiles with few copy number aberrations. Gene expression analysis showed SCCOHT tumors have a unique gene expression profile unlike that of most common epithelial ovarian carcinomas. Dysregulated cell cycle control, DNA repair, DNA damage-response, nucleosome assembly, neurogenesis and nervous system development were all characteristic of SCCOHT tumors. Sequencing of DNA from SCCOHT patients and cell lines revealed germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 79% (19/24) of SCCOHT patients in addition to SMARCA4 protein loss in 84% (16/19) of SCCOHT tumors, but in only 0.4% (2/485) of other primary ovarian tumors. Ongoing studies are now focusing on identifying treatments for SCCOHT based on therapeutic vulnerabilities conferred by ubiquitous inactivating mutations in SMARCA4 in addition to gene and protein expression data. Our characterization of the molecular landscape of SCCOHT and the breakthrough identification of inactivating SMARCA4 mutations in almost all cases of SCCOHT offers the first significant insight into the molecular pathogenesis of this disease. The loss of SMARCA4 protein is a highly sensitive and specific marker of the disease, highlighting its potential role as a diagnostic marker, and offers the opportunity for genetic testing of family members at risk. Outstanding questions remain about the role of SMARCA4 loss in the biology, histogenesis, diagnosis, and treatment of SCCOHT.
ContributorsRamos, Pilar (Author) / Anderson, Karen (Thesis advisor) / Trent, Jeffrey (Committee member) / Kusumi, Kenro (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2014
Description
Skeletal muscle injury may occur from repetitive short bursts of biomechanical strain that impair muscle function. Alternatively, variations of biomechanical strain such as those held for long-duration are used by clinicians to repair muscle and restore its function. Fibroblasts embedded within the unifying connective tissue of skeletal muscle experience these multiple and diverse mechanical stimuli and respond by secreting cytokines. Cytokines direct all stages of muscle regeneration including myoblasts differentiation, fusion to form myotubes, and myotube functionality. To examine how fibroblasts respond to variations in mechanical strain that may affect juxtapose muscle, a myofascial junction was bioengineered that examined the interaction between the two cell types. Fibroblasts were experimentally shown to increase myoblast differentiation, and fibroblast biomechanical strain mediated the extent to which differentiation occurred. Intereleukin-6 is a strain-regulated cytokine secreted by fibroblasts was determined to be necessary for fibroblast-mediated myoblast differentiation. Myotubes differentiated in the presence of strained fibroblasts express greater number of acetylcholine receptors, greater acetylcholine receptor sizes, and modified to be more or less sensitive to acetylcholine-induced contraction. This study provides direct evidence that strained and non-strained fibroblasts can serve as a vehicle to modify myoblast differentiation and myotube functionality. Further understanding the mechanisms regulating these processes may lead to clinical interventions that include strain-activated cellular therapies and bioengineered cell engraftment for mediating the regeneration and function of muscle in vivo.
ContributorsHicks, Michael (Author) / Standley, Paul R (Thesis advisor) / Rawls, Jeffrey (Committee member) / Lake, Douglas (Committee member) / Hinrichs, Richard (Committee member) / Arizona State University (Publisher)
Created2014
Description
Osteosarcoma is the most common bone cancer in children and adolescents. Patients with metastatic osteosarcoma are typically refractory to treatment. Numerous lines of evidence suggest that cytotoxic T-lymphocytes (CTL) limit the development of metastatic osteosarcoma. I have investigated the role of Programmed Death Receptor-1 (PD-1) in limiting the efficacy of immune mediated control of metastatic osteosarcoma. I show that human metastatic, but not primary, osteosarcoma tumors express the ligand for PD-1 (PD-L1) and that tumor infiltrating CTL express PD-1, suggesting this pathway may limit CTL control of metastatic osteosarcoma in patients. PD-L1 is also expressed on the K7M2 osteosarcoma tumor cell line that establishes metastases in mice, and PD-1 is expressed on tumor infiltrating CTL during disease progression. Blockade of PD-1/PD-L1 interactions dramatically improves the function of osteosarcoma-reactive CTL in vitro and in vivo, and results in decreased tumor burden and increased survival in the K7M2 mouse model of metastatic osteosarcoma. My results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma should be pursued as a therapeutic strategy. However, PD-1/PD-L1 blockade treated mice still succumb to disease due to selection of PD-L1 mAb resistant tumor cells via up-regulation of other co-inhibitory T cell receptors. Combinational α-CTLA-4 and α-PD-L1 blockade treated mice were able to completely eradicate metastatic osteosarcoma, and generate immunity to disease. These results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma, although improves survival, may lead to tumor resistance, requiring combinational immunotherapies to combat and eradicate disease.
ContributorsLussier, Danielle (Author) / Blattman, Joseph N. (Thesis advisor) / Anderson, Karen (Committee member) / Goldstein, Elliott (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2015
Description
Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme that catalyzes disulfide bond formation by oxidizing two free sulfhydryl groups. QSOX1 consists of a thioredoxin (Trx) and an ERV (essential for respiration and viability)/ALR (augmenter of liver regeneration) domain which each contain CxxC motifs that work to bind to substrates and shuttle electrons to a flavin adenine dinucleotide (FAD) cofactor that accepts the electrons and reduces molecular oxygen to hydrogen peroxide. Investigation of the role of QSOX1 in cancer progression started when it was found at higher abundance in pancreatic ductal adenocarcinoma (PDA) patient plasma compared to healthy normal donor plasma. Increased expression in QSOX1 has been further identified in breast, lung, kidney, prostate, and other cancers. QSOX1 expression is associated with cell proliferation and invasion in vitro and tumor growth in vivo. Additionally, the enzymatic activity of QSOX1 in the extracellular matrix (ECM) is important for cell invasion in vitro. Small molecule inhibitors of QSOX1 have been shown to have antitumorigenic properties in vitro and in vivo. It was hypothesized that monoclonal antibodies (mAbs) against QSOX1 would inhibit cell invasion in vitro. In this work, mice were immunized with eukaryotic-derived rQSOX1 for generation of hybridomas. Hundreds of hybridoma clones were screened by enzyme-linked immunosorbent assay (ELISA) and a fluorescent QSOX1 activity assay. Multiple rounds of subcloning and screening identified 2F1.14 and 3A10.6 as mAbs of interest. The genes for the variable regions of the antibodies were rescued and sequenced. The sequences were aligned with the variable region sequences of another published αQSOX1 mAb scFv492.1. 2F1.14 inhibits the enzymatic activity of QSOX1 by binding to the active site of QSOX1, which was determined by epitope mapping against mutants of QSOX1 that contained mutations in the active site. 3A10.6 did not appear to inhibit the function of QSOX1 in the activity assay; however, it, along with 2F1.14, suppressed tumor invasion in a 3D invasion model. These findings support the developing idea that QSOX1 is a viable target for cancer treatment because targeted inhibition of QSOX1 extracellularly reduced invasive activity. The mAbs and rQSOX1 variants produced here can serve as tools in furthering the characterization of QSOX1 and its role in cancer.
ContributorsKoelbel, Calvin John (Author) / Lake, Douglas (Thesis advisor) / Chen, Qiang "Shawn" (Committee member) / Ho, Thai (Committee member) / Arizona State University (Publisher)
Created2019
Description
In species with highly heteromorphic sex chromosomes, the degradation of one of the sex chromosomes can result in unequal gene expression between the sexes (e.g., between XX females and XY males) and between the sex chromosomes and the autosomes. Dosage compensation is a process whereby genes on the sex chromosomes achieve equal gene expression which prevents deleterious side effects from having too much or too little expression of genes on sex chromsomes. The green anole is part of a group of species that recently underwent an adaptive radiation. The green anole has XX/XY sex determination, but the content of the X chromosome and its evolution have not been described. Given its status as a model species, better understanding the green anole genome could reveal insights into other species. Genomic analyses are crucial for a comprehensive picture of sex chromosome differentiation and dosage compensation, in addition to understanding speciation.
In order to address this, multiple comparative genomics and bioinformatics analyses were conducted to elucidate patterns of evolution in the green anole and across multiple anole species. Comparative genomics analyses were used to infer additional X-linked loci in the green anole, RNAseq data from male and female samples were anayzed to quantify patterns of sex-biased gene expression across the genome, and the extent of dosage compensation on the anole X chromosome was characterized, providing evidence that the sex chromosomes in the green anole are dosage compensated.
In addition, X-linked genes have a lower ratio of nonsynonymous to synonymous substitution rates than the autosomes when compared to other Anolis species, and pairwise rates of evolution in genes across the anole genome were analyzed. To conduct this analysis a new pipeline was created for filtering alignments and performing batch calculations for whole genome coding sequences. This pipeline has been made publicly available.
In order to address this, multiple comparative genomics and bioinformatics analyses were conducted to elucidate patterns of evolution in the green anole and across multiple anole species. Comparative genomics analyses were used to infer additional X-linked loci in the green anole, RNAseq data from male and female samples were anayzed to quantify patterns of sex-biased gene expression across the genome, and the extent of dosage compensation on the anole X chromosome was characterized, providing evidence that the sex chromosomes in the green anole are dosage compensated.
In addition, X-linked genes have a lower ratio of nonsynonymous to synonymous substitution rates than the autosomes when compared to other Anolis species, and pairwise rates of evolution in genes across the anole genome were analyzed. To conduct this analysis a new pipeline was created for filtering alignments and performing batch calculations for whole genome coding sequences. This pipeline has been made publicly available.
ContributorsRupp, Shawn Michael (Author) / Wilson Sayres, Melissa A (Thesis advisor) / Kusumi, Kenro (Committee member) / DeNardo, Dale (Committee member) / Arizona State University (Publisher)
Created2016
Description
Acute Myeloid Leukemia (AML) is a disease that occurs when genomic changes alter expression of key genes in myeloid blood cells. These changes cause them to resume an undifferentiated state, proliferate, and maintain growth throughout the body. AML is commonly treated with chemotherapy, but recent efforts to reduce therapy toxicity have focused on drugs that specifically target and inhibit protein products of the cancer’s aberrantly expressed genes. This method has proved difficult for some proteins because of structural challenges or mutations that confer resistance to therapy. One potential method of targeted therapy that circumvents these issues is the use of small molecules that stabilize DNA secondary structures called G-quadruplexes. G-quadruplexes are present in the promoter region of many potential oncogenes and have regulatory roles in their transcription. This study analyzes the therapeutic potential of the compound GQC-05 in AML. This compound was shown in vitro to bind and stabilize the regulatory G-quadruplex in the MYC oncogene, which is commonly misregulated in AML. Through qPCR and western blot analysis, a GQC-05 mediated downregulation of MYC mRNA and protein was observed in AML cell lines with high MYC expression. In addition, GQC-05 is able to reduce cell viability through induction of apoptosis in sensitive AML cell lines. Concurrent treatment of AML cell lines with GQC-05 and the MYC inhibitor (+)JQ1 showed an antagonistic effect, indicating potential competition in the silencing of MYC. However, GQC-05 is not able to reduce MYC expression significantly enough to induce apoptosis in less sensitive AML cell lines. This resistance may be due to the cells’ lack of dependence on other potential GQC-05 targets that may help upregulate MYC or stabilize its protein product. Three such genes identified by RNA-seq analysis of GQC-05 treated cells are NOTCH1, PIM1, and RHOU. These results indicate that the use of small molecules to target the MYC promoter G-quadruplex is a viable potential therapy for AML. They also support a novel mechanism for targeting other potentially key genetic drivers in AML and lay the groundwork for advances in treatment of other cancers driven by G-quadruplex regulated oncogenes.
ContributorsTurnidge, Megan (Author) / Lake, Douglas (Thesis advisor) / Kim, Suwon (Committee member) / Azorsa, David (Committee member) / Arizona State University (Publisher)
Created2017
Description
IOsteosarcoma is the most common bone cancer and typically affects patients in the second decade of life. Current treatment methods have not proven effective for treating reoccurring or metastatic osteosarcoma (mOS) given the 5-year survival rate of 15-30%. Previous work showed that using the immune system to fight the cancer significantly improved survival of mOS in mice, but approximately 40-50% of treated mice still succumbed to disease. To further improve immunotherapy, I analyzed immune cells in the tumor bed and observed high numbers of a rare T cell subtype: CD4hiCD8αhi, or double positive (DP), T cells. While previous literature found mature DP T cells in chronic diseases, the associations and functions of this rare T cell subtype varied between studies and were unknown for mOS. Controlling for age, chronicity of disease, and environmental exposure, I found DP T cells composed a higher percentage of T cells in the cancer as tumor burden increased. I then tested whether the DP cells were pro- or anti-tumor. I found that DP cells produced the cytokines IFNγ and IL-2 when exhaustion was overcome. They also expressed FasL for cytotoxic function, although the target is unknown. These findings suggest DP T cells have multifunctionality, which could be advantageous when responding to high antigen load.
II
Course-based undergraduate research experiences (CUREs) offer students opportunities to engage in critical thinking and problem solving. However, quantitating the impact that incorporating research into undergraduate courses has on student learning has been difficult since most CUREs lack a comparable traditional course as a control. Because the overall class structure remained unaltered when our upper division immunology course transitioned to a CURE class, we realized retrospectively that we were in a unique position to quantitate the impact of incorporating research on student performance. I then analyzed the summative assessments used to assess student learning and found that students in the CURE format class performed significantly better on quizzes, exams, and reports. There were no significant differences in academic levels, degree programs, or grade point averages, suggesting improved performance was due to increased engagement of students in research.
ContributorsAppel, Nicole (Author) / Blattman, Joseph (Thesis advisor) / Anderson, Karen (Committee member) / Lake, Douglas (Committee member) / Hingorani, Pooja (Committee member) / Arizona State University (Publisher)
Created2022
Description
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) that emerged from a zoonotic host at the end of 2019 and caused a public health crisis. In this collection of studies, Nicotiana benthamiana plants are used to set the foundation for producing monoclonal antibodies (mAbs) with homogeneous glycosylation to neutralize SARS-CoV-2 and potentially address the immunopathology often observed with severe COVID-19. Specifically, a mAb against the human interleukin (IL)-6 receptor (sarilumab) was generated and evaluated in vitro for its potential to reduce IL-6 signaling that has been shown to be associated with more severe cases of COVID-19. Furthermore, multiple mAbs that bind to the receptor-binding domain (RBD) of SARS-CoV-2 and efficiently neutralize the virus were developed using plant-based expression. Several of these mAbs are from different classes of RBD-binding mAbs that have distinct binding sites from one another. Several mAbs from different classes showed synergy in neutralizing the ancestral strain of SARS-CoV-2 and a smaller subset showed synergy when tested against the highly mutated Omicron (B.1.1.529) variant. Of interest, a novel RBD-binding mAb, termed 11D7, that was raised against the ancestral strain and derived from a hybridoma, appears to have an epitope on the RBD that contributes more synergy to a mAb combination that efficiently neutralizes the B.1.1.529 variant of SARS-CoV-2. This epitope was partially mapped by competitive binding and shows that it overlaps with another known antibody that binds a cryptic, distal epitope, away from the receptor binding site, giving insight into the potential mechanism by which 11D7 neutralizes SARS-CoV-2, as well as potentially allowing it to resist SARS-CoV-2 immune evasion more efficiently. Furthermore, this mAb carries a highly homogeneous glycan pattern when expressed in N. benthamiana, that may contribute to enhanced effector function and provides a tool to elucidate the precise role of crystallizable fragment (Fc)-mediated protection in SARS-CoV-2 infection. Ultimately, these studies provide evidence of the utility of plant-made mAbs to be used as cocktail members, giving clarity to the use of less potent mAbs as valuable cocktail components which will spur further investigations into how mAbs with unique epitopes work together to efficiently neutralize SARS-CoV-2.
ContributorsJugler, Collin (Author) / Chen, Qiang (Thesis advisor) / Lake, Douglas (Committee member) / Steele, Kelly (Committee member) / Mason, Hugh (Committee member) / Arizona State University (Publisher)
Created2022
Description
Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that represents the ancient fusion of two major thiol-disulfide oxidoreductase gene families: thioredoxin and ERV. QSOX1 was first linked with cancer after being identified as overexpressed in pancreatic ductal adenocarcinoma (but not in adjacent normal ductal epithelia, infiltrating lymphocytes, or chronic pancreatitis). QSOX1 overexpression has been confirmed in a number of other histological tumor types, such as breast, lung, kidney, prostate, and others. Expression of QSOX1 supports a proliferative and invasive phenotype in tumor cells, and its enzymatic activity is critical for promoting an invasive phenotype. An in vivo tumor growth study utilizing the pancreatic tumor cell line MIAPaCa-2 containing a QSOX1-silencing shRNA construct revealed that QSOX1 expression supports a proliferative phenotype. These preliminary studies suggest that suppressing the enzymatic activity of QSOX1 could represent a novel therapeutic strategy to inhibit proliferation and invasion of malignant neoplasms.
The goal of this research was to identify and characterize biologically active small molecule inhibitors for QSOX1. Chemical inhibition of QSOX1 enzymatic activity was hypothesized to reduce growth and invasion of tumor cells. Recombinant QSOX1 was screened against libraries of small molecules using an enzymatic activity assay to identify potential QSOX1 inhibitors. Two lead QSOX1 inhibitors were confirmed, 2-phenyl-1, 2-benzisoselenazol-3-one (ebselen), and 3-methoxy-n-[4-(1 pyrrolidinyl)phenyl]benzamide. The biological activity of these compounds is consistent with QSOX1 knockdown in tumor cell lines, reducing growth and invasion in vitro. Treatment of tumor cells with these compounds also resulted in specific ECM defects, a phenotype associated with QSOX1 knockdown. Additionally, these compounds were shown to be active in pancreatic and renal cancer xenografts, reducing tumor growth with daily treatment. For ebselen, the molecular mechanism of inhibition was determined using a combination of biochemical and mass spectrometric techniques. The results obtained in these studies provide proof-of-principle that targeting QSOX1 enzymatic activity with chemical compounds represents a novel potential therapeutic avenue worthy of further investigation in cancer. Additionally, the utility of these small molecules as chemical probes will yield future insight into the general biology of QSOX1, including the identification of novel substrates of QSOX1.
The goal of this research was to identify and characterize biologically active small molecule inhibitors for QSOX1. Chemical inhibition of QSOX1 enzymatic activity was hypothesized to reduce growth and invasion of tumor cells. Recombinant QSOX1 was screened against libraries of small molecules using an enzymatic activity assay to identify potential QSOX1 inhibitors. Two lead QSOX1 inhibitors were confirmed, 2-phenyl-1, 2-benzisoselenazol-3-one (ebselen), and 3-methoxy-n-[4-(1 pyrrolidinyl)phenyl]benzamide. The biological activity of these compounds is consistent with QSOX1 knockdown in tumor cell lines, reducing growth and invasion in vitro. Treatment of tumor cells with these compounds also resulted in specific ECM defects, a phenotype associated with QSOX1 knockdown. Additionally, these compounds were shown to be active in pancreatic and renal cancer xenografts, reducing tumor growth with daily treatment. For ebselen, the molecular mechanism of inhibition was determined using a combination of biochemical and mass spectrometric techniques. The results obtained in these studies provide proof-of-principle that targeting QSOX1 enzymatic activity with chemical compounds represents a novel potential therapeutic avenue worthy of further investigation in cancer. Additionally, the utility of these small molecules as chemical probes will yield future insight into the general biology of QSOX1, including the identification of novel substrates of QSOX1.
ContributorsHanavan, Paul D (Author) / Lake, Douglas (Thesis advisor) / LaBaer, Joshua (Committee member) / Mangone, Marco (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2015
Description
Quiescin Sulfhydryl Oxidase 1 (QSOX1) generates disulfide bonds in its client substrates via oxidation of free thiols. Localized to the Golgi and secreted, QSOX1 helps to fold proteins into their active form. Early work with QSOX1 in cancer began with the identification of a peptide from the long form of QSOX1 in plasma from patients with pancreatic ductal adenocarcinoma. Subsequent work confirmed the overexpression of QSOX1 in numerous cancers in addition to pancreatic, including those originating in the breast, lung, brain, and kidney. For my work, I decided to answer the question, “How does inhibition of QSOX1 effect the cancer phenotype?” To answer this I sought to fulfill the following goals A) determine the overexpression parameters of QSOX1 in cancer, B) identify QSOX1 small molecule inhibitors and their effect on the cancer phenotype, and C) determine potential biological effects of QSOX1 in cancer. Antibodies raised against rQSOX1 or a peptide from QSOX1-L were used to probe cancer cells of various origins for QSOX1 expression. High-throughput screening was utilized to identify 3-methoxy-n-[4(1pyrrolidinyl)phenyl]benzamide (SBI-183) as a lead inhibitor of QSOX1 enzymatic activity. Characterization of SBI-183 activity on various tumor cell lines revealed inhibition of viability and invasion in vitro, and inhibition of growth, invasion, and metastasis in vivo, a phenotype that was consistent with QSOX1 shKnockdown cells. Subsequent work identified 3,4,5-trimethoxy-N-[4-(1-pyrrolidinyl)phenyl]benzamide (SPX-009) as an SBI-183 analog with stronger inhibition of QSOX1 enzymatic activity, resulting in a more potent reduction in tumor invasion in vitro. Additional work with QSOX1 shKnockdown and Knockout (KO) cell lines confirmed current literature that QSOX1 is biologically active in modulation of the ECM. These results provide evidence for the master regulatory role of QSOX1 in cancer, making it an attractive chemotherapeutic target. Additionally, the small molecules identified here may prove to be useful probes in further elucidation of QSOX1 tumor biology and biomarker discovery.
ContributorsFifield, Amber (Author) / Lake, Douglas (Thesis advisor) / Ho, Thai (Committee member) / Rawls, Jeffery (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2020