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The Pennsylvania State University The Pennsylvania State University The Graduate School Department of Neurosurgery EXOSOMES AND THEIR IMPLICATIONS IN GLIOBLASTOMA A Dissertation in Biomedical Sciences by Oliver D. Mrowczynski 2018 Oliver D. Mrowczynski Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2018 ii The dissertation of Oliver D. Mrowczynski was reviewed and approved* by the following: James R. Connor University Distinguished Professor Vice-Chair of the Department of Neurosurgery Dissertation Advisor Chair of Committee Jennifer W. Baccon Professor and Chair Department of Pathology, Northeast Ohio Medical University Chair, Department of Pathology and Laboratory Medicine, Akron Children’s Hospital Barbara A. Miller Professor of the Department of Pediatrics Achuthamangalam B. Madhankumar Assistant Professor of the Department of Neurosurgery Brad E. Zacharia Assistant Professor of the Department of Neurosurgery Special Member Jong K. Yun Associate Professor of the Department of Pharmacology Ralph L. Keil Chair, Biomedical Sciences Graduate Program Associate Professor of Biochemistry and Molecular Biology *Signatures are on file in the Graduate School iii ABSTRACT Exosomes are 20-100 nm cellular derived vesicles that when discovered, were initially thought to be a form of cellular recycling of intracellular contents. More recently, these vesicles are under study for their purported significant roles in intercellular communication in both healthy and diseased states. Exosomes are secreted by all cancer types and have a major impact on the tumor microenvironment. Exosomes contain protected intracellular content, including DNA, RNA, and proteins that can be transferred to recipient cells. This transfer subsequently leads to enhanced tumorigenic properties including angiogenesis, cancer progression, and therapeutic resistance. Genetic components of the cell of origin can be included in the secreted exosomes. The presence of genetic material could serve as a biomarker for the presence of mutations in tumors leading to modification of treatment strategies. In the last decade, exosomes have been identified as having major implications in many aspects of medicine and tumor biology and appear to be primed to take a critical position in cancer diagnosis, prognosis, and treatment. The first aspect of my thesis studies the effects of changes in genotype on exosome phenotype by studying neuroblastoma cancer and hemochromatosis mutations. Neuroblastoma is the third most common childhood cancer, and timely diagnosis and sensitive therapeutic monitoring remain major challenges. Tumor progression and recurrence is common in advanced stages with little understanding of mechanisms. Although exosomes have been demonstrated to contribute to the oncogenic effect on the surrounding tumor environment and also mediate resistance to therapy, the effect of genotype on exosomal phenotype had not been explored. I interrogated exosomes from human neuroblastoma cells that express wild-type or mutant forms of the HFE gene. HFE, one of the most common autosomal recessive polymorphism in the Caucasian population, originally associated with hemochromatosis, has also been associated with increased tumor burden, therapeutic resistance boost, and negative impact on patient survival. iv Herein, I demonstrated that changes in genotype cause major differences in the molecular and functional properties of exosomes; specifically, HFE mutant derived exosomes have increased expression of proteins relating to invasion, angiogenesis, and cancer therapeutic resistance. HFE mutant derived exosomes were also shown to transfer this cargo to recipient cells and cause an increased oncogenic functionality in those recipient cells. This has major implications because it allows clinicians to profile and stratify cancer patients with HFE mutations and adapt therapeutic strategies to provide patients with the optimal survival outcome. The second aspect of my thesis assessed the effects of radiation on exosome function and profile. Radiation therapy is essential in the arsenal of cancer treatment and is utilized in the therapeutic regimen of more than 50% of all cancer patients. Unfortunately, many aggressive malignancies may become resistant to radiation over time, rendering treatment futile. This mechanism of acquired radiation resistance is not understood. I investigated the hypothesis that acquired radiation resistance may occur through cellular communication via exosomes. Three aspects were analyzed: 1) exosome function, 2) exosome profile, and 3) exosome uptake/blockade. Radiation-derived exosomes increased cellular proliferation and radiation resistance in recipient tumor cells in vitro in cell culture. Furthermore, radiation-derived exosomes increased tumor burden and decreased survival in vivo in a murine model of glioblastoma. The mechanism underlying this phenomenon is that radiation-derived exosomes exhibit specific miRNA, mRNA, and protein expression changes favoring a resistant/proliferative profile. Radiation-derived exosomes upregulate oncogenic miR-889, CCND1, ANXA2, DERL1, WWC1, NPM1, SCD, ACTG1, FUT11, VAMP8, ZFR, DNM2, CISD1, RPL15, PPIC, and proteins involved in the proteasome, Notch, Jak-STAT, and cell cycle signaling pathways. Radiation-derived exosomes also downregulate tumor-suppressive miR-516, miR-365, TPM1, STAT4, LRRFIP1, TSPAN5, and CGGBP1. Ingenuity pathway analysis revealed the top upregulated networks included cell growth, cell cycle, and cell survival. The top increased v upstream regulator was the MYC oncogene. Upregulation of Dynamin 2 correlated with increased uptake of radiation-derived exosomes. In addition to inducing changes in recipient cells that promote a cancerous phenotype, I evaluated exosome blockade as a potential therapeutic. Heparin and simvastatin blocked uptake of radiation-derived exosomes in recipient cells and inhibited induction of cellular proliferation and radiation resistance, both in vitro and in vivo. In conclusion, I provide a novel exosome-based mechanism that may underlie acquired radiation resistance in patients harboring malignant cancers. Furthermore, I elucidate key factors carried by exosomes that may lead to tumor recurrence and subsequent therapeutic resistance. I also show the potential for advancement of cancer treatment, or understanding existing treatments, whose mechanism may be through exosome inhibition. By interrogating this mechanism, I ultimately aim to pave the way for the development and implementation of targeted exosome blocking agents to inhibit the acquired radiation resistance that inevitably leads to tumor recurrence and the devastating prognosis that follows. The last aspect of my thesis assessed the effects of glioma cell stemness on exosome phenotype. One reason proposed for the ineffectiveness of the current therapeutic regimen of glioblastoma is glioma stem cells (GSCs). We investigated the hypothesis that the communication of GSCs to their microenvironment through exosomes is a key factor to the enhanced tumor burden and the development of resistance to therapeutics in glioblastoma. Two properties of exosomes were analyzed: 1) exosome function and 2) exosome profile. Exosomes secreted by patient derived-glioma stem cells (GSC-exosomes) increased cellular proliferation, radiation resistance, temozolomide resistance, and doxorubicin resistance. We further profiled the GSC- exosomes to elucidate the underlying mechanism of this phenomenon. Profiling showed specific changes to RNA and protein favoring therapeutic resistance and cellular proliferation. GSC exosomes have increased expression of proteins involved in radiation and chemotherapeutic resistance (E.g. CDK4 and Notch), cellular proliferation (E.g. Cyclin B1 and Cyclin D2), vi angiogenesis (E.g. VEGF-A and EGFR), glioma cell stemness and de-differentiation (E.g. EPHA2, Cathepsin B), and cell invasion and metastasis (E.g. ITGA3, COL4A2). The results of our study provide a novel exosome-based mechanism that may underlie the aggressiveness of glioma cancer stem cells. Furthermore, we elucidate key factors carried by glioma stem cell derived exosomes that may lead to enhanced therapeutic resistance and increase in tumor burden. Together, these data demonstrate the impact that exosomes have on multiple aspects of tumor biology. Exosomes are affected due to genotype, and thus genotype must be taken into consideration for cancer patient stratification. Exosomes are also are critical for radiation resistance and are reprogrammed due to radiation. Glioma stem cells secrete exosomes which have increased oncogenic contents and have an enhanced functional impact on recipient cancer cells. My studies have shown that treatment with heparin and simvastatin inhibits uptake of these cancer-derived exosomes which may lead to enhanced therapeutic efficacy in the cancer patient population. My thesis also suggests that our current therapies to treat glioblastoma may be ineffective due to exosomes secreted by glioma stem cells in conjunction with exosomes secreted by glioma cells stressed with radiation. These secreted exosomes confer therapeutic resistance and may lead to subsequent tumor recurrence in the glioblastoma patient population. The results of my thesis offer direct recommendation for clinical studies that suggests utilizing our standard therapies in combination with exosome uptake inhibitors may lead to optimal patient outcomes. vii TABLE OF CONTENTS List of Figures .........................................................................................................................
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