<p>mRNA localization is emerging as a critical cellular mechanism for the spatiotemporal regulation of protein expression and serves important roles in oogenesis, embryogenesis, cell fate specification, and synapse formation. Signal sequence-encoding mRNAs are localized to the endoplasmic reticulum (ER) membrane by either of two mechanisms, a canonical mechanism of translation on ER-bound ribosomes (signal recognition particle pathway), or a poorly understood direct ER anchoring mechanism. In this study, we identify that the ER integral membrane proteins function as RNA-binding proteins and play important roles in the direct mRNA anchoring to the ER. We report that one of the ER integral membrane RNA-binding protein, AEG-1 (astrocyte elevated gene-1), functions in the direct ER anchoring and translational regulation of mRNAs encoding endomembrane transmembrane proteins. HITS-CLIP and PAR-CLIP analyses of the AEG-1 mRNA interactome of human hepatocellular carcinoma cells revealed a high enrichment for mRNAs encoding endomembrane organelle proteins, most notably encoding transmembrane proteins. AEG-1 binding sites were highly enriched in the coding sequence and displayed a signature cluster enrichment downstream of encoded transmembrane domains. In overexpression and knockdown models, AEG-1 expression markedly regulates translational efficiency and protein functions of two of its bound transcripts, MDR1 and NPC1. This study reveals a molecular mechanism for the selective localization of mRNAs to the ER and identifies a novel post-transcriptional gene regulation function for AEG-1 in membrane protein expression.</p> / Dissertation

Glioblastomas continue to carry poor prognoses for patients despite advances in surgical, chemotherapeutic, and radiation regimens. One feature of glioblastoma associated with poor prognosis is the degree of hypoxia and elevated expression levels of hypoxia-inducible factor-1 á (HIF-1á). HIF-1á expression allows metabolic adaptation to low oxygen availability, partly through upregulation of vascular endothelial growth factor (VEGF) and increased tumor angiogenesis as well as induction of anaerobic glycolysis. In this study, we demonstrate an induced level of astrocyte-elevated gene-1 (AEG-1) by hypoxia in glioblastoma cells. AEG-1 has the capacity to promote anchorage-independent growth and cooperates with Ha-ras in malignant transformation. In addition, AEG-1 was recently demonstrated to serve as an oncogene and can induce angiogenesis and autophagy in glioblastoma. Results from in vitro studies show that hypoxic induction of AEG-1 is dependent on HIF-1á stabilization during hypoxia and that phosphatidylinositol 3-kinase (PI3K) inhibition abrogates AEG-1 induction during hypoxia through loss of HIF-1á stability. Furthermore, we show that AEG-1 is induced by glucose deprivation and that prevention of intracellular reactive oxygen species (ROS) production prevents this induction. Additionally, AEG-1 knockdown results in increased ROS production and increased glucose deprivation-induced cytotoxicity, whereas AEG-1 overexpression prevents ROS production and decreases glucose deprivation-induced cytotoxicity, indicating that AEG-1 induction is necessary for cells to survive this type of cell stress. From studies examining the expression of enzymes involved in glucose metabolism, we demonstrate that AEG-1 alters the tumor metabolic profile in a partially 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK)-dependent manner. Moreover, glycolytic inhibition modulates the metabolic effects induced by AEG-1, and AEG-1 knockdown reduces the growth and alters the metabolic phenotype of glioblastoma subcutaneous xenografts. These observations link AEG-1 overexpression observed in glioblastoma with hypoxia and glucose metabolic signaling, and targeting these physiological pathways may lead to therapeutic advances in the treatment of glioblastoma in the future. Recent studies have reported the detection of the human neurotropic virus, JC Virus (JCV), in a significant population of brain tumors, including medulloblastomas. Accordingly, expression of the JCV early protein, T-antigen, which has transforming activity in cell culture and in transgenic mice, results in the development of a broad range of tumors of neural crest and glial origin. Evidently, the association of T-antigen with a range of tumor-suppressor proteins, including p53 and pRb, and signaling molecules, such as â-catenin and IRS-1, play a role in the oncogenic function of JCV T-antigen. We demonstrate that T-antigen expression is suppressed by glucose deprivation in medulloblastoma cells that endogenously express T-antigen. Mechanistic studies indicate that glucose deprivation-mediated suppression of T-antigen is partly influenced by AMPK, a critical sensor of the AMP/ATP ratio in cells. We have found that AMPK activation inhibits T-antigen expression, whereas AMPK inhibition prevents glucose deprivation-mediated T-antigen suppression. In addition, glucose deprivation-induced cell cycle arrest in the G1 phase is blocked with AMPK inhibition, which also prevents T-antigen downregulation. Furthermore, T-antigen-expressing medulloblastoma cells, as compared to those which do not express T-antigen, exhibit less G1 arrest and an increased percentage of cells in the G2 phase of the cell cycle during glucose deprivation. On a functional level, T-antigen downregulation is partially dependent on ROS production during glucose deprivation. Additionally, studies indicate that T-antigen prevents ROS induction, loss of ATP production, and cytotoxicity induced by glucose deprivation. We have also found that T-antigen is downregulated by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the pentose phosphate inhibitors, 6-aminonicotinamde (6-AN) and oxythiamine (OT). Enzyme expression studies also indicate that T-antigen upregulates the expression of the pentose phosphate enzyme, transaldolase-1 (TALDO1), demonstrating a potential link between T-antigen and glucose metabolic regulation. These studies highlight the potential involvement of JCV T-antigen in the proliferation and metabolic phenotype of medulloblastoma and may enhance our understanding of the role of viral proteins in tumor glycolytic metabolism, thus implicating these proteins as potential targets for the treatment of virus-associated tumors. / Biomedical Neuroscience

First identified over a decade ago, Astrocyte Elevated Gene-1 (AEG-1) has been studied extensively due to early reports of its overexpression in various cancer cell lines. Research groups all over the globe including our own have since identified AEG-1 overexpression in cancers of diverse lineages including cancers of the liver, colon, skin, prostate, breast, lung, esophagus, neurons and neuronal glia as compared to matched normal tissue. A comprehensive and convincing body of data currently points to AEG-1 as an essential component, critical to the progression and perhaps onset of cancer. AEG-1 is a potent activator of multiple pro-tumorigenic signal transduction pathways such as mitogen-activated protein extracellular kinase (MEK)/ extracellular signal-regulated kinase (ERK), phosphotidyl-inositol-3-kinase (PI3K)/Akt/mTOR, NF-κB and Wnt/β-catenin pathway. In addition, studies show that AEG-1 not only alters global gene and protein expression profiles, it also modulates fundamental intracellular processes, such as transcription, translation and RNA interference in cancer cells most likely by functioning as a scaffold protein. The mechanisms by which AEG-1 is overexpressed in cancer have been studied extensively and it is clear that multiple layers of regulation including genomic amplification, transcriptional, posttranscriptional, and posttranslational controls are involved however; the mechanism by which AEG 1 itself induces its oncogenic effects is still poorly understood. Just as questions remain about the exact role of AEG-1 in carcinogenesis, very little is known about the role of AEG-1 in regulating normal physiological functions in the liver. With the help of the Massey Cancer Center Transgenic/Knockout Mouse Core, our lab has successfully created a germline-AEG-1 knockout mouse (AEG-1-/-) as a model to interrogate AEG-1 function in vivo. Here I present the insights gained from efforts to analyze this novel AEG-1-/- mouse model. Aspects of the physiological functions of AEG-1 will be covered in chapter two wherein details of the characterization of the AEG-1-/- mouse are described including the role of AEG-1 in lipid metabolism. Chapter three discusses novel discoveries about the specific role of AEG-1 in mediating hepatocarcinogenesis by modulating NF-κB, a critical inflammatory pathway. First identified over a decade ago, Astrocyte Elevated Gene-1 (AEG-1) has been studied extensively due to early reports of its overexpression in various cancer cell lines. Research groups all over the globe including our own have since identified AEG-1 overexpression in cancers of diverse lineages including cancers of the liver, colon, skin, prostate, breast, lung, esophagus, neurons and neuronal glia as compared to matched normal tissue. A comprehensive and convincing body of data currently points to AEG-1 as an essential component, critical to the progression and perhaps onset of cancer. AEG-1 is a potent activator of multiple pro-tumorigenic signal transduction pathways such as mitogen-activated protein extracellular kinase (MEK)/ extracellular signal-regulated kinase (ERK), phosphotidyl-inositol-3-kinase (PI3K)/Akt/mTOR, NF-κB and Wnt/β-catenin pathway. In addition, studies show that AEG-1 not only alters global gene and protein expression profiles, it also modulates fundamental intracellular processes, such as transcription, translation and RNA interference in cancer cells most likely by functioning as a scaffold protein. The mechanisms by which AEG-1 is overexpressed in cancer have been studied extensively and it is clear that multiple layers of regulation including genomic amplification, transcriptional, posttranscriptional, and posttranslational controls are involved however; the mechanism by which AEG 1 itself induces its oncogenic effects is still poorly understood. Just as questions remain about the exact role of AEG-1 in carcinogenesis, very little is known about the role of AEG-1 in regulating normal physiological functions in the liver. With the help of the Massey Cancer Center Transgenic/Knockout Mouse Core, our lab has successfully created a germline-AEG-1 knockout mouse (AEG-1-/-) as a model to interrogate AEG-1 function in vivo. Here I present the insights gained from efforts to analyze this novel AEG-1-/- mouse model. Aspects of the physiological functions of AEG-1 will be covered in chapter two wherein details of the characterization of the AEG-1-/- mouse are described including the role of AEG-1 in lipid metabolism. Chapter three discusses novel discoveries about the specific role of AEG-1 in mediating hepatocarcinogenesis by modulating NF-κB, a critical inflammatory pathway.