25 February 2016
Angiogenesis expands the vascular network during normal development and in response to angiogenic stress. Dysregulation of this dynamic process contributes to the pathogenesis of many diseases including retinopathies. The α2β1 integrin, a collagen and laminin receptor which is linked to risk of vascular retinopathy, plays an important yet incompletely understood role in angiogenesis. In this dissertation, I employ multidisciplinary approaches to examine the function of this integrin during both pathological and developmental angiogenesis in the retina. The major goal is to contribute clinically relevant knowledge through mechanistic investigation of the link between α2β1 integrin in vascular retinopathies and careful exploration of this integrinâs role in angiogenesis. The central questions addressed in this thesis are, 1) does the α2β1 integrin contribute to the progression of retinopathies, and through what mechanism? And, 2) how does α2β1 integrin interface with the major angiogenesis pathways, and does that explain the divergent effects of α2-integrin deletion in different vascular beds? Using the oxygen-induced retinopathy (OIR) model for retinopathy of prematurity (ROP) on wild type and α2-null mice, I elucidated the role of α2β1 integrin in both endothelial cells as well as in the retinal microenvironment. I uncover a novel, potentially mechanistically important role for the integrin in regulating retinal MÃ¼ller cell function. To clarify the role of α2β1 integrin in angiogenesis, I use in vitro, in vivo and in silico methods to characterize wild type and α2-null mice during postnatal development of the retinal vasculature. I develop a hybrid mathematical model to simulate cell signaling and vascular morphology in the developing postnatal murine retina. Using this model, I study how the VEGF-notch signaling system directs the development of morphological features including, retinal vascularization, plexus density, and plexus irregularity. I also use the model to predict how crosstalk to the VEGF-Notch axis from other angiogenic signals affects vascular phenotypes. Finally, I use the computational model as a platform for evaluating proposed signaling relationships between α2β1 integrin and the VEGF-Notch axis and present a molecular model which may explain how α2-integrin deletion causes disparate vascular phenotypes in different vascular microenvironments.
Youngblood, Victoria Marie
30 March 2016
Dysregulation of receptor tyrosine kinases (RTKs) contributes to cellular transformation and cancer progression by disrupting key metabolic signaling pathways. The EPHA2 RTK is overexpressed in aggressive forms of breast cancer, including the HER2+ subtype, and correlates with poor prognosis. However, the role of EPHA2 in tumor metabolism remains unexplored. This thesis sought to determine the mechanisms by which EPHA2 ligand-independent signaling promotes tumorigenesis in the absence of its prototypic ligand, ephrin-A1. We integrated in vivo and in vitro models of HER2-overexpressing breast cancer to demonstrate that loss of ephrin-A1 leads to upregulated glutamine metabolism and lipid accumulation that enhanced tumor growth. Global metabolic profiling of ephrin-A1-null, HER2-overexpressing mammary tumors revealed a significant increase in glutaminolysis, a critical metabolic pathway that generates intermediates for lipogenesis. Pharmacologic inhibition of glutaminase activity reduced tumor growth in both ephrin-A1-depleted and EPHA2-overexpressing tumor allografts in vivo. Mechanistically, we show that the enhanced proliferation and glutaminolysis in the absence of ephrin-A1 was attributed to increased RhoA-dependent glutaminase activity. EPHA2 depletion or pharmacologic inhibition of Rho, glutaminase, or fatty acid synthase abrogated the proliferative effects of ephrin-A1 knockdown. Together, these findings highlight a novel, unsuspected connection between the Ephrin-A1/EPHA2 signaling axis and tumor metabolism, and suggest potential new therapeutic targets in cancer subtypes exhibiting glutamine dependency.
Austin, David C.
31 March 2016
Benign prostatic hyperplasia (BPH) is a common, progressive chronic disease. Inflammation is associated with prostatic enlargement and resistance to 5?-reductase inhibitor (5ARI) therapy. Activation of the nuclear factor-kappa B (NF-?B) pathway is linked to both inflammation and ligand-independent prostate cancer progression. Most patients initially respond to 5ARI therapy; however, failure is common. To address why patients fail therapy we used transition zone tissue samples from patients with a wide range of American Urological Association symptom score (AUASS) from incidental BPH in patients treated for low grade, localized peripheral zone prostate cancer to advanced disease requiring surgical intervention. NF-?B activation and androgen receptor variant (AR-V) expression were quantified. To further investigate these pathways, human prostatic stromal and epithelial cell lines were transduced with constitutively active or kinase dead forms of IKK2 to regulate canonical NF-?B activity, AR-FL, and AR-variant 7 (AR-V7). We determined that canonical NF-?B signaling was found to be upregulated in late versus early stage BPH. Elevated expression of AR-V7 was found in advanced BPH samples. Expression of AR-V7 significantly correlated with the patient AUASS. Forced activation of canonical NF-?B in human prostatic epithelial and stromal cells resulted in elevated expression of both AR-FL and AR-V7, with concomitant ligand-independent activation of AR reporters. Activation of NF-?B and over expression of AR-V7 in human prostatic epithelial cells maintained cell viability in the face of 5ARI treatment. To understand why NF-?B and AR-V7 maintained viability within 5ARI treatment we examined the levels of 5?-reductase enzymes (SRD5A1, SRD5A2, SRD5A3). We determined that SRD5A2 is upregulated in more advanced BPH. SRD5A2 was significantly associated with AUASS and patients on a 5ARI. AR-FL and AR-V7 expression increased SRD5A2 expression whereas forced NF-?B activation increased all SRD5A isoforms. In summary, activation of NF-?B and AR-V7 in the prostate is associated with increased disease severity. Increased BPH severity in patients correlates with SRD5A2 expression. De novo synthesis of androgens and AR-V7 expression is inducible in human prostate cells by forced activation of NF-?B. NF-?B and AR-V7 upregulate SRD5A2 resulting in resistance to 5ARI treatment, suggesting a potential mechanism by which patients may become resistant to 5ARI therapy.
Identification and Characterization of Neural-like Cancer Stem Cells in Salivary Adenoid Cystic CarcinomaPanaccione, Alexander Colin 04 April 2016 (has links)
Salivary adenoid cystic carcinoma (ACC) is prone to perineural invasion, late recurrence, and distal metastases, with 20-year survival of only 10%. Research defining new targets in ACC has lagged largely due to a dearth of in vitro models. In this thesis, a neurogenic gene signature intrinsic to ACC was characterized revealing genes involved in maintenance, differentiation, and function of non-cancerous neural crest stem cells, including TrkC and SOX10. Analyses of gene expression across tumor types revealed that melanoma, neuroblastoma, glioblastoma, and basal-like breast cancer expressed SOX10 and several SOX10 co-expressed genes, suggesting that tumors derived from cells originating in the neural crest may contain similar populations of stem-like cells. Examination of TrkC signaling revealed that TrkC supported cell migration, invasion, and ACC tumor growth, and that ACC cells produced the TrkC ligand. Optimization of newly-designed culturing techniques allowed for the first time establishment of ACC cell cultures from xenograft and primary ACC specimens. Gene expression analysis revealed that CD133 was co-expressed with SOX10, and remarkably, it marked a subpopulation of ACC cells that preferentially expressed NOTCH1 and SOX10, formed spheroids, and initiated tumors in nude mice. Depletion of NOTCH1 or pharmaceutical inhibition of Notch signaling depleted CD133+ cells, sensitized CD133+ cells to radiation, and suppressed spheroidogenesis and xenograft tumor formation. Optimization of culture techniques provides a framework for future experimentation, and identification of a stem-like subpopulation in ACC suggests that targeting signaling pathways critical for stem cell survival or behavior may provide a new avenue to ACC therapy.
Tseng, Roger Sean
Human papillomaviruses (HPV) are small non-enveloped viruses that infect basal cells. Most HPV infections are mild and develop warts at the site of infection. However, some high-risk serotypes of HPV are able to promote cancer formation. Serotypes 16 and 18 are responsible for the majority of cervical cancer cases . Its early proteins E6 and E7 promote oncogenesis by facilitating the acquisition of 7 hallmark traits necessary for cancer: constant signaling for proliferation, insensitivity to growth suppressors, evasion of apoptosis, limitless replicative potential, angiogenesis, immune evasion, and metastasis [1, 65, 68, 72, 76, 78, 79, 81, 82, 83, 84]. In addition to E6 and E7, specific conditions of an HPV infection seem to increase cancer incidence. Among these conditions are infection at the cervix's transformation zone, HPV genome integration with host chromosomes, inflammation and the presence of estrogen [1, 60, 61, 62, 63, 64, 69, 70, 71]. Estrogen's role in cervical cancer is not well understood. It is possible that it plays a role in the transcription of oncogenes by activating ERα and subsequently activating Sp1 . Specifically, the Sp1 binding site is conserved and necessary for VEGF and hTERT expression [65, 79].
Varga, Matthew Gordon
12 August 2016
CANCER BIOLOGY The Role of Toll-Like Receptor 9 in Helicobacter pylori-Mediated Inflammation and Carcinogenesis Matthew Gordon Varga Dissertation under the direction of Dr. Richard M. Peek Jr., MD Helicobacter pylori is the strongest identified risk factor for gastric cancer, the third most common cause of cancer-related death worldwide. The H. pylori cag pathogenicity island encodes a type IV secretion system (T4SS), which translocates the pro-inflammatory protein CagA into host cells. Although T4SSs are ubiquitous and facilitate transport of diverse effectors from bacterial cells to the eukaryotic host, only two bacterial species have been shown to translocate DNA into eukaryotic host cells. Furthermore, in DNA-translocating T4SSs, the only proteins transported via the T4SS are proteins associated with the process of DNA transfer. Toll-like receptor 9 is an endosome bound, innate immune receptor that detects hypomethylated CpG DNA motifs. We now demonstrate that the H. pylori cag T4SS is required for TLR9 activation and that H. pylori DNA is actively translocated by the cag T4SS to engage this host receptor. Genetic deficiency of Tlr9 augments the intensity of IL-17-driven immune responses to H. pylori in vivo, suggesting that engagement of TLR9 by bacterial DNA leads to suppression of inflammation, which may accommodate long-term interactions between pathogenic H. pylori and the gastric epithelium. In support of this hypothesis, analysis of a human population at increased risk for gastric cancer revealed that levels of H. pylori-mediated TLR9 activation were directly related to the severity of cancer risk. These results demonstrate the ever-increasing versatility of T4SS machineries and their involvement in promoting pathogen persistence and modulating disease outcomes.
27 June 2016
Î³Ketoaldehydes are generated by free radical peroxidation and cyclooxygenation of phospholipid-esterified arachidonic acid. Î³Ketoaldehydes covalently bind to protein lysine residues and is emerging as a mechanistic link between pathogenic reactive oxygen species and disease progression. However, the questions of whether covalent modification of proteins by Î³KA are subject to genetic regulation and the identity of Î³KA-modified proteins remain unclear. This dissertation shows that Nrf2 and Nox2 are key regulators of Î³KA modification in pulmonary tissue. The identity of these proteins were analyzed by LC-MS following immunoaffinity purification of Î³KA-modified proteins. Gene ontology analysis revealed that proteins in numerous cellular pathways are susceptible to Î³KA modification. Although cells tolerate basal levels of modification, exceeding them induces apoptosis. Prominent modification was observed in a murine model of radiation-induced pulmonary fibrosis and in idiopathic pulmonary fibrosis (IPF) patients, two diseases considered to be promoted by gene-regulated oxidant stress. This dissertation found an abundance of Î³KA modified protein in human IPF compared to control lung tissue, identified collagen 1Î±1 as one of the highly adducted proteins, and demonstrates that Î³KA modification impairs MMP1 mediated degradation of collagen. Based on these results the current hypothesis is that Î³KA modification is a hitherto unrecognized sequelae that contributes to radiation-induced pulmonary injury and IPF.
26 July 2016
Exosomes are extracellular vesicles that carry a variety of RNA and protein cargos, including growth factors, transmembrane proteins, angiogenic factors, proteinases, and microRNA. However, the mechanisms that regulate exosome secretion are still poorly understood. This dissertation work shows that the tumor-overexpressed cytoskeletal protein cortactin promotes exosome secretion. Knockdown or overexpression of cortactin in head and neck squamous cell carcinoma, fibrosarcoma, and breast cancer cells leads to a corresponding decrease or increase in exosome secretion. However, cortactin knockdown does not affect cargo sorting or exosome biogenesis. Rescue experiments carried out by adding purified exosomes back to cortactin-knockdown cells suggest that exosome secretion may account for many functions of cortactin in tumor aggressiveness. Furthermore, live imaging revealed that cortactin controls both trafficking and plasma membrane docking of multivesicular endosomes. Mechanistically, interaction of cortactin with Arp2/3 complex and branched actin is critical for exosome secretion. Also, cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin multivesicular endosome docking sites along with exosome secretion. Overall, this dissertation identifies molecular and cellular mechanisms important for exosome secretion.
Hanke, Neale T.
Primarily defined by their antigen presenting function, dendritic cells (DC) are equipped with the unique ability to initiate and regulate immune responses. A less conventional characteristic of DC has been highlighted more recently: their capability to directly kill tumor cells when appropriately activated. The main objectives of the study presented herein were to analyze the molecular regulation of DC cytotoxic activity and to determine how the tumoricidal potential of DC may influence their cardinal antigen presenting function. To address these questions, DC were generated from myeloid precursors with either IL-4 (IL-4 DC) or IL-15 (IL-15 DC). We demonstrate that IL-4 and IL-15 DC exhibit similar iNOS-dependent tumor killing activity when activated with the toll-like receptor (TLR)-4 agonist LPS. However, stimulation with interferon (IFN)-γ selectively induces iNOS-dependent cytotoxic activity of IL-4 but not IL-15 DC. Possible differences in the signaling pathways controlling iNOS expression in these two DC populations were then examined. In both IL-4 and IL-15 DC, LPS initially activates NF-κB, followed by secondary activation of components of the ISGF3 transcription factor. Using inhibitors and knockout mice we established that disruption of the NF-κB or ISGF3 signaling axes impaired LPS-induced iNOS expression in IL-15 DC with little to no effect in IL-4 DC. A distinct and separate JAK-STAT pathway is required for iNOS induction in IL-4 DC activated with IFN-γ. IL-15 DC express high levels of PIAS1 and phosphorylated STAT-3 which act as independent inhibitors of iNOS expression upon stimulation with IFN-γ. Inhibiting PIAS1 with silencing RNA (siRNA) along with STAT-3 inhibition or knockdown restores iNOS expression and the tumor killing activity of IL-15 DC stimulated with IFN-γ. We further established that following culture with cancer cells, DC endowed with cytolytic activity are more efficient at presenting antigens to specific T lymphocytes compared to their counterparts generated from iNOS^(-/-) mice, which are significantly impaired in their tumoricidal function. This indicates that the capability of DC to present tumor-specific antigens may be contingent upon induction of their cytotoxic activity.
Bates, Andreia LaShonne
18 March 2015
CANCER BIOLOGY Roles of Fibroblast MMP2 in Breast to Lung Metastasis Andreia L. Bates Dissertation under the direction of Barbara Fingleton, PhD Breast cancer five-year survival rates decrease from 99% for patients with local disease to 25% for those with distant metastases. The potential for tumor cells to proliferate in a foreign microenvironment and develop into overt metastases is mediated through interactions with stromal cells at the secondary site. Understanding the mechanisms underlying tumor cell cooperation with the stromal milieu to support the outgrowth of metastases is essential for increasing patient survival. Matrix metalloproteinases (MMPs), including MMP2, are associated with metastatic progression. Preliminary studies found that loss of host MMP2 reduced the proliferation of experimental metastases in the lungs of mice, and identified fibroblasts in control tumor-bearing lungs as the major source of MMP2. In vitro, spheroidal mammary tumor growth was increased by co-culture with control fibroblasts isolated from tumor-bearing lungs but not when fibroblasts with Mmp2 knockdown were used. This result suggested that MMP2 was responsible for a tumor-proliferative, activated fibroblast phenotype. The studies within this dissertation employ a combination of in vitro, in vivo, and correlation analyses using a human data set to investigate how MMP2 potentiates breast tumor outgrowth in the lungs. In vitro, exogenous active enzyme increased tumor cell proliferation in 3D but not 2D. Knockdown of Mmp2 in fibroblasts attenuated expression of two markers of activation (á-smooth muscle actin and vimentin). Additionally, Mmp2 knockdown fibroblasts showed significantly decreased expression of the matrix transcripts collagen I, collagen IV and fibronectin. Active TGFâ-1 was sufficient to rescue the MMP2-dependent collagen I and IV expression, while MMP2-induced collagen expression was blocked with addition of TGFâ-1 neutralizing antibody. Gene expression data in stromal cells of human breast cancers revealed that MMP2 expression is also positively correlated with activation and matrix transcripts. A model is presented whereby MMP2 production in tumor fibroblasts is important for the activity of TGFâ-1 cytokine and subsequent activation of fibroblasts to a matrix-producing, proliferation-supportive phenotype. Overall, these studies reveal a previously undefined role for MMP2 in metastatic outgrowth mediated by fibroblasts, and extends the mechanisms by which MMPs contribute to tumor progression. Approved___________________________ Date_______ Barbara Fingleton, PhD
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