Breast cancer has the highest incidence rate in women, accounting for more than 22% of all cancers and possessing a strong disposition to metastasize to bone. These skeletal metastases become a significant cause of morbidity and mortality in patients with the primary symptom being pain. Pain is a major concern in determining a patient’s quality of life and there have been many attempts to understand and control bone pain with little success. Previous studies have shown that glutamate plays a role in bone cancer pain, with an excess in free glutamate able to cause pain either directly through excitotoxic pathways or indirectly though the dysregulation of osteoclasts and osteoblasts, causing bone dysregulation. TRPV-1 receptors have also has been implicated in the mechanisms of bone cancer pain, as osteoclasts release protons during bone remodeling which can elicit a TRPV-1-related nociceptive response from neurons in the surrounding periosteum. Capsazepine was identified during a high throughput screen of 30,000 compounds to be a potent inhibitor of breast cancer cell-mediated glutamate release, a neurotransmitter with known associations in neural signaling, bone homeostasis, and pain. Capsazepine also has antagonistic effects on transient receptor potential vanilloid type 1 (TRPV-1) receptors which act as key players in both heat and vanilloid-induced nociception. These findings suggest that Capsazepine may provide a multi-site effect for the treatment of cancer-induced bone pain. An animal model of breast cancer-induced bone pain involved intrafemorally injecting MDA-MB-231 cancer cells to measure pain. Behavioural tests are then performed measuring dynamic weight bearing and paw withdrawal thresholds. These measurements are used to demonstrate both movement-evoked and spontaneous pain-related behaviour of the affected limb. Using Capsazepine, we demonstrate a dose-dependent attenuation of pain behaviour in vivo, while confirming tumour presence using immunohistochemistry (IHC). We show that TRPV-1 and glutamate play an important role in the onset and severity of bone cancer pain and blocking these pain pathways provide relief from pain commonly associated with cancer in the bone. / Thesis / Master of Health Sciences (MSc)

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Huntington's disease (HD) is an inherited autosomal-dominant neurodegenerative disease caused by the abnormal expansion of CAG repeats in exon 1 of the huntingtin gene located on chromosome 4. This disease is characterized by the premature loss of medium spiny neurons in the striatum and behavioural deficits that typically manifest at middle-age. Despite the identification of its cause decades ago, there is still no disease modifying treatment available for HD patients. Current evidence indicates that exacerbated glutamate signalling in the striatum plays a key role in the pathophysiology of HD. Within the striatum, metabotropic glutamate receptor (mGluR) 2/3 are predominantly expressed on presynaptic terminals, whereas mGluR5 is predominantly localized to postsynaptic terminals. Here, we show that both the activation of mGluR2/3 and the inhibition of mGluR5 can improve HD symptoms in the zQ175 HD mouse model. Specifically, treating zQ175 HD mice with either the mGluR2/3 agonist LY379268 or the mGluR5 negative allosteric modulator (NAM) CTEP rescues motor deficits, reduces mutant huntingtin aggregate formation, improves neuronal survival and alleviates microglia activation. We also provide evidence that shows sex can influence the progression of HD symptoms and the efficacy of therapeutic agents. We found that chronic administration of LY379268 differentially activated and inactivated cell signalling pathways in male and female zQ175 mice. Furthermore, female zQ175 mice required a longer treatment duration with CTEP than male mice to show improvement in their rotarod performance. Using FDNQ175 mice, a newer HD mouse model derived from the zQ175 line, we demonstrated that female FDNQ175 mice were less susceptible to decline in limb function than male mice but showed higher levels of insoluble mutant huntingtin aggregates at a younger age.

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Cancer-induced pain (CIP) is a debilitating condition that accompanies late-stage cancer for the majority of patients. The work presented in this dissertation addresses the multifaceted role of glutamate in cancer cell-induced pain signalling and provides several potential therapeutic directions. Several cell types, including breast cancer cells and microglia, release glutamate via the system xC- antiporter. To limit the excitotoxic tendency of breast cancer cells to release glutamate in excess, we first indirectly inhibited xCT, the active subunit of system xC-, with the TrkA inhibitor AG879. We demonstrated that the system xC- antiporter is functionally influenced by the actions of nerve growth factor on its cognate receptor, TrkA, and that inhibiting this complex reduced CIP via downstream actions on xCT. Co-culture studies then demonstrated the direct effect of glutamate released by wildtype MDA-MB-231 carcinoma cells on microglial activation, as well as functional system xC- activity, while knockdown of xCT in MDA-MB-231 cells mitigated microglial activation and cystine uptake. Blockade of system xC- with sulfasalazine attenuated nociception in an immunocompetent murine model of CIP and inhibited tumour-induced microglial activation in the dorsal horn of the spinal cord. Finally, tumour-induced nociceptive behaviours appeared to progress in parallel with microglial activation in the hippocampus, and ablating microglia delayed the onset and severity of tumour-induced nociceptive behaviours, confirming that microglia are implicated in CIP and regional microglia are influenced by this pain. This is the first experimental evidence to demonstrate the effects of peripheral tumour on hippocampal microglial activation in relation to cancer-related nociception. These data collectively demonstrate that the system xC- antiporter is functionally implicated in CIP and may be particularly relevant to pain progression through spinal microglia. Upregulated xCT in chronically activated microglia may be one pathway to central glutamate cytotoxicity. Therefore, microglial xCT may therefore be a valuable target for mitigating CIP. / Thesis / Doctor of Philosophy (Medical Science) / Cancer-induced pain (CIP) is a debilitating condition that accompanies late-stage metastatic cancer. Clinically, achieving analgesia often comes at the expense of patients’ quality of life, as current therapeutics fail to adequately manage this pain and induce dose-dependent side effects. Cancer cells secrete excess amounts of glutamate, a signalling molecule involved in CIP, which can activate immune cells called microglia within the spinal cord. Mice that demonstrate tumour-induced pain exhibit an amplified immune response that manifests through the activation pattern and quantity of microglia within the spinal cord, as well as brain regions implicated in pain and distress. Pharmacologically blocking glutamate release from cancer cells limits this pain response, in addition to several physiological indicators of pain, including microglial activation in the central nervous system. Changes in microglia-related glutamate signalling may reflect the emotional problems reported by patients with CIP. Better understanding the mechanisms of CIP will help generate more comprehensive treatment approaches.

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.