Glioblastoma Multiforme (GBM) is the most common and malignant form of adult brain cancer, with 95% of patients succumbing to the disease within 5 years of diagnosis. An important contributing factor to this poor prognosis is upregulation of the transmembrane protein system xc- (SXC) found on GBM cells. Approximately 50% of GBM patients have tumors with upregulated levels of SXC, and these patients experience faster disease progression than patients with tumors expressing moderate levels of SXC. SXC is a sodium-independent antiporter and is comprised of a light chain catalytic subunit (xCT) bound to a heavy chain regulatory subunit (4f2hc/CD98) via a disulfide bond. The xCT subunit is responsible for the equimolar exchange of extracellular cystine for intracellular glutamate. Clinical studies have shown areas immediately surrounding the tumor, known as the peritumoral region, reach glutamate concentrations over 100 times that of the normal brain, creating an excitotoxic environment in which neurons cannot survive. In addition to neuronal excitotoxicity, excess glutamate release has also been shown to promote GBM cell invasion, as well as contributing to the clinical presentation of seizures in patients. Moreover, cystine is a component of the antioxidant glutathione, which confers protection to the cells from alkylating therapeutics such as temozolomide (TMZ).
In an effort to identify novel targets that regulate SXC function, I investigated the relationship between SXC and two signaling molecules known to promote GBM progression: CD44 and the epidermal growth factor receptor (EGFR). I experimentally manipulated the CD44-hyaluronic acid (HA) interaction and EGFR to determine if these two signaling molecules were involved in regulating SXC expression and function in two patient-derived GBM cell lines. Experimental data led me to conclude that the tumorigenic potential conferred to GBM cells by CD44 is not related to an interaction with SXC. However, I found that knocking down EGFR led to a significant reduction in SXC expression. These findings are important to the field, as combinatorial therapies become more actively pursued in clinical trials. Inhibition of EGFR may provide quality of life benefits to patients who suffer from tumor-associated epilepsy through downregulating xCT-mediated glutamate release. / Doctor of Philosophy / Glioblastoma multiforme (GBM) is an advanced and aggressive form of brain cancer. Incidence of this disease in the United States of America is approximately 3.19 per 100,000 individuals, which translates to more than 13,000 expected annual diagnoses. These tumors arise from genetic mutations that instruct cells to replicate and migrate abnormally. Despite an aggressive medical armamentarium that includes maximal surgical resection, chemotherapy, and radiation, GBM patients have an expected survival period of 12-15 months after diagnosis.
Previous studies have shown that approximately 50% of GBM patients have unusually high expression levels of the System xc- (SXC) protein. SXC is a protein transporter located at the membrane of GBM cells, and facilitates the exchange of the excitatory neurotransmitter glutamate for the amino acid dimer cystine. SXC exports glutamate out of the tumor cell, where it can then bind to glutamate receptors on surrounding neurons. In the brain, the concentration of extracellular glutamate must be tightly regulated to prevent hyperexcitability of neurons, which may lead to cell death and the induction of seizures. In patients whose tumors highly express SXC, studies have shown that glutamate levels can rise to concentrations over 100 times greater than the levels seen in normal brain tissue. Additionally, glutamate has been shown to stimulate GBM cells to migrate within the brain and establish secondary tumor sites.
The medical and scientific community is justifiably interested in discovering novel methods for regulating or inhibiting SXC-mediated glutamate release. While SXC inhibitors have been identified, clinical studies have determined they are not appropriate for the clinical treatment of GBM. Thus the focus of this project was to identify novel molecular regulators of SXC. To that end, I explored two signaling molecules that are known to promote GBM pathogenesis: CD44 and the epidermal growth factor receptor (EGFR). I found no evidence to support a role for CD44 in regulating SXC in GBM. However, I was able to determine, through genetic and pharmacologic manipulation of patient-derived GBM cells, that EGFR regulates SXC expression and function. The results of these experiments confirmed EGFR as a key signaling protein involved in orchestrating SXC-mediated glutamate release, and may inform future clinical studies investigating combinatorial therapies for GBM patients.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/112876 |
| Date | 21 June 2021 |
| Creators | Martin, Joelle Dominique |
| Contributors | Graduate School, Sontheimer, Harald W., Gourdie, Robert G., Munson, Jennifer M., Olsen, Michelle Lynne |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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