Serotonin has been widely explored in the brain. Recently, there have been new findings on how serotonin works in the periphery. Serotonin is introduced to the periphery by the enterochromaffin cells and metabolized by the liver and lung. Studies have shown that serotonin plays a role in controlling lung cancer. However, the mechanism by which it initiates tumor formation has not been fully explored. Cell viability was measured in several lung adenocarcinoma cell lines treated with serotonin to study this effect. In GFP-labelled cells, fluorescence intensity was measured for quantification of cell viability. Our data showed an overall increase in viability when serotonin concentration was increased, which is significant because it shows that serotonin affects lung cancer progression. We will look at how serotonin works on tumor cells compared to endothelial cells and its effect on immune system activation. This study hopes to inspire future anti-angiogenesis and immunotherapy studies of lung cancer by understanding this interaction.

The structural and functional abnormalities of the tumor vasculature generate regions of elevated interstitial fluid pressure and aberrant flow shear stress within the tumor microenvironment. While research has shown that the hydrodynamics of the tumor vasculature reduce transport and uptake of therapeutic agents, the underlying mechanisms by which fluid forces regulate vascular organization are not well known. Understanding the reciprocal interaction between tumor and endothelial cells to mediate angiogenesis, and the role of flow shear stress on this process, may offer insight into the design of improved therapeutic strategies to control vascularized tumors. Instrumental to this is the development of physiologically relevant models that enable tumor-endothelial co-culture under dynamic conditions. By integrating tissue-engineering strategies with cancer biology, micro-scale fluid mechanics, and optical flow diagnostics, the goal of this research was to develop a 3D in vitro microfluidic culture model to investigate tumor-endothelial cross talk under physiologically relevant flow shear stress. This objective was motivated by early findings demonstrating a contact-independent, paracrine-mediated mechanism by which endothelial cells enhance tumor-expressed angiogenic factors during 2D, static co-culture. The 3D tumor vascular model consists of a central microchannel embedded within a type I collagen hydrogel, through which a range of normal (4 dyn/cm^2), low (1 dyn/cm^2) and high (10 dyn/cm^2) microvascular wall shear stresses (WSS) were introduced. Endothelial cells lining the microchannel lumen form a confluent endothelium across which soluble growth factors are exchanged with tumor cells in the gel. Microscopic particle image velocimetry ("-PIV) was integrated within the model to enable noninvasive optical measurement of velocity profiles and quantification of WSS, which were then correlated with angiogenic potential. Results demonstrate that endothelial permeability decreases as a function of increasing WSS, while co-culture with tumor cells increases permeability. This response is likely due to shear stress-mediated endothelial cell alignment and tumor-VEGF-induced permeability. In addition, high WSS (10 dyn/cm^2) significantly down-regulates tumor-expressed angiogenic factors, suggesting flow shear stress-mediates endothelial cross talk with surrounding tumor cells. Collectively, this research demonstrates the utility of the 3D in vitro microfluidic culture model as a versatile platform for elucidating the role of tumor-relevant hydrodynamic stress on cellular response. / Ph. D.

Epithelial cells that lose attachment to the extracellular matrix (ECM) undergo a specialized form of apoptosis called anoikis. Anoikis has an important role in preventing oncogenesis, particularly metastasis, by eliminating cells that lack proper ECM cues. The basis of anoikis resistance remains to be determined and to date has not been linked to alterations in expression or activity of previously identified anoikis effector genes. Here, I utilized two different screening strategies to identify novel anoikis effector genes and miRNAs in order to gain a deeper understanding of anoikis and the potential mechanisms of anoikis resistance in cancer. Using large-scale RNA interference (RNAi) screening, I found that KDM3A, a histone H3 lysine 9 (H3K9) mono- and di-demethylase plays a pivotal role in anoikis induction. In attached breast epithelial cells, KDM3A expression is maintained at low levels by integrin signaling. Following detachment, integrin signaling is decreased resulting in increased KDM3A expression. RNAi-mediated knockdown of KDM3A substantially reduces apoptosis following detachment and, conversely, ectopic expression of KDM3A induces cell death in attached cells. I found that KDM3A promotes anoikis through transcriptional activation of BNIP3 and BNIP3L, which encode pro-apoptotic proteins. Using mouse models of breast cancer metastasis I show that knockdown of Kdm3a enhances metastatic potential. Finally, I find defective KDM3A expression in human breast cancer cell lines and tumors. Collectively, my results reveal a novel transcriptional regulatory program that mediates anoikis. Next, I sought to discover miRNAs involved in anoikis by investigated changes in miRNA expression during anoikis using small RNA sequencing technology. Through this approach I discovered that miR-203 is an anoikis effector miRNA that is also highly down-regulated in invasive breast cancer cells. In breast epithelial cells, miR-203 is induced upon the loss of ECM attachment and inhibition of miR-203 activity leads to a resistance to anoikis. I utilized a dual functional- and expression- based RNA sequencing approach and found that miR-203 directly targets a network of pro-survival genes to induce cell death upon detachment. Finally, I found that the loss of miR-203 in invasive breast cancer leads to the elevation of several anoikis-related pro-survival target genes to contribute to anoikis resistance. Taken together, my studies reveal novel pathways through which cell death is induced upon detachment from the ECM and provide insight into potential mechanisms of anoikis resistance in cancer.

<p>Breast cancer is the most common cancer among Canadian women with one in nine women expected to develop breast cancer in their lifetime. Until recently these breast tumors were thought to be a homogeneous cell population. Recent studies have shown that breast tumors contain a rare cell type termed breast tumor initiating cells (TICs) or cancer stem cells (CSCs) with the ability to elicit new tumor growth and metastases. These TICs exist apex of a tumor cell hierarchy and give rise to more TICs and non-tumorigenic cells. Traditionally, drugs were developed to target the highly proliferative cells population resulting in a decrease in tumor volume. However, these therapies spare the TICs, which results in tumor relapse demonstrating the need for new drugs that target the TICs. Because in cancer, mutated protein kinases are the controllers of cell proliferation, invasion and metastasis, they have become a target for drug development. Inhibition of these kinases could lead to the identification of compounds that selectively target breast TICs. Using mammary tumors from cancer prone mice propagated as non-adherent tumorspheres (TMS), which contain a high fraction of breast TICs and the same conditions to propagate the non-transformed mouse mammary epithelial stem and progenitor cells (MESC), as non-adherent mammospheres (MMS) a 240-kinase inhibitor library was screened using an AlamarBlue proliferation assay. Twenty percent of the compounds resulted in 75% decrease in proliferation of TMS derived cells and some of which were TMS-selective. Sunitinib, a multi-targeted kinase inhibitor, was one of the selective compounds identified and when administered to mice with subcutaneous mammary tumors resulted in tumor shrinkage. This was accompanied by an increase in apoptotic cells, decrease in proliferating cells and tumor vasculature, and a change in tumor morphology and composition. These findings show the efficacy of Sunitinib in shrinking mouse mammary tumors and suggest a potential use of Sunitinib for treatment of breast cancer.</p> / Master of Science (MSc)

<p><strong>A growing body of evidence suggests that most human tumors, including those of the breast, are organized as cellular hierarchies. Positioned at the apex of these hierarchies are tumor-initiating cells (TICs), which are capable of limitless self-renewal and also differentiate, to give rise to various populations of non-tumorigenic cells that make up the bulk of the tumor. Importantly, recent findings have demonstrated that TICs are refractory to current best practice therapies, and thus likely account for high rates of tumor recurrence following remission. Therefore, it will likely be important to identify novel means of targeting TICs in order to achieve durable cancer cures.</strong></p> <p><strong>Using a highly sensitive transplantation assay, our laboratory previously showed that mammary tumors arising in various strains of transgenic mice comprise a very high fraction of TICs, and that when cells from these tumors are propagated in serum-free medium as tumorspheres, the high frequency of TICs is maintained. We therefore sought to use mouse mammary tumorspheres as an <em>in vitro</em> system with which to identify TIC-targeted agents and carried out a high-throughput screen of nearly 32,000 small molecules. To eliminate compounds showing general toxicity, we employed mouse mammospheres, which primarily comprise normal mammary epithelial stem and progenitor cells, in a secondary screen. Using this platform, we identified a small molecule that selectively targeted tumorsphere-derived cells <em>in vitro</em> and led to tumor growth arrest and tumor cell death <em>in vivo</em>. This study illustrates the utility of mouse models and high throughput screening to identify compounds which may target TICs but spare untransformed stem cells.</strong></p> / Master of Science (MSc)

<p>Brain metastases are most common in adults suffering from lung cancer, predicting uniformly poor patient outcome and short survival time. Despite their frequency and severity, very little is known about the tumorigenesis of brain metastases. Previously developed primary brain tumour-initiating cell (BTIC) models were used to determine the presence of a stem-like population in brain metastases from the lung. Use of clinical samples and the NCI-H1915 cell line allowed for the development of useful strategies for study of brain metastasis.</p> <p>The sphere formation capacity and expression of known BTIC markers in brain metastases was suggestive of a self-renewing population. Differentiation studies demonstrated that neither clinical samples nor NCI-H1915 cells had neural lineage potential. Intracranial xenotransplant of clinical samples and NCI-H1915 cells into NOD-SCID mice led to formation of multiple focal masses throughout the ventricles; the tumours were also serially transplantable, further implicating a TIC population. Of known BTIC markers, only CD15 expression levels and patterns were similar enough in clinical samples and NCI-H1915 cells to warrant prospective sorting experiments in the cell line. Use of CD15 failed to identify a CSC or TIC population in NCI-H1915 cells.</p> <p>These findings suggest that a TIC population is present in brain metastases; however, this remains to be identified. It is recommended that due to the limitations of cell surface markers, the study of brain metastasis should use a selective gene expression approach, in order to target genes and pathways essential to metastasis. It was shown that NCI-H1915 cells could be useful for such an approach, studying the effects on proliferation, sphere formation, and tumour formation capacity of brain metastases from the lung. Further study using this model could ultimately lead to the disruption of pathways essential to the metastatic process, transforming a uniformly fatal disease into a more localized and treatable one.</p> / Master of Science (MSc)

<p>Chondrosarcoma is a unique type of bone cancer in that it does not respond to chemotherapy or radiation therapy, and therefore many affected patients die from metastatic disease. Metastasis has been correlated to upregulation of the matrix metalloproteinase (MMP) family of proteases, which can degrade a wide range of extracellular components. MMP2 is an enzyme secreted from the cell and degrades extracellular gelatin, as denatured collagen. ETV5 is a transcription factor which has shown to be overexpressed in multiple types of invasive tumours. The regulatory relationship between ETV5 and MMP2 has not been studied in chondrosarcoma. We hypothesized that ETV5 regulates MMP2 in human chondrosarcoma with the protease acting as a downstream effector. We have determined that both ETV5 and MMP2 are upregulated in the KC human chondrosarcoma cell line. Gene knockdown of ETV5 in KC cells resulted in a reduction in MMP2 mRNA expression as well as decreased protein production, and significantly decreased MMP2 gelatinase activity. When ETV5 is overexpressed, MMP2 expression is upregulated at the RNA and protein levels. Data from our bone resorption studies revealed that when a matrix metalloproteinase 2 inhibitor is added to the KC cell growth media, collagen released from bone chips decreased significantly. Upon histological examination, bone chips incubated with KC cells and the MMP2 inhibitor showed less bone resorption. Analyses of patient cell lines show similar expression profiles of ETV5 and MMP2 as the KC cell line. ETV5 knockdown (KD) in the patient cell lines also showed a decrease in MMP2 expression, similar to the KC cells, suggesting that the ETV5-MMP2 pathway has clinical importance. This data advocates that ETV5 has a significant role in regulating MMP2 expression and activity, and bone resorption in human chondrosarcoma, and thus may be a targetable effector of the metastatic cascade in this cancer.</p> / Master of Science (MSc)

<p>The POZ-ZF transcription factor ZBTB4 was initially identified due to its sequence homology to the dual-specificity DNA-binding transcription factor Kaiso. Subsequent characterization of ZBTB4 revealed that it is also a dual-specificity DNA-binding protein; it recognizes a specific oligonucleotide sequence C^T/_CGCCATC, coined the <strong>Z</strong>BTB<strong>4</strong> <strong>B</strong>inding <strong>S</strong>equence (Z4BS) as well as methylated CpG-dinucleotides. Interestingly, ZBTB4 also binds to the highly similar consensus <strong>K</strong>aiso <strong>B</strong>inding <strong>S</strong>ite (KBS) <em>in vitro</em>.</p> <p>ZBTB4 is misexpressed in cancer, and follows a stage-specific pattern of expression in breast carcinoma tissues; low ZBTB4 levels are found in late stages while high ZBTB4 expression is detected in early stages of disease progression. Ongoing studies have begun to elucidate the molecular interactions that mediate ZBTB4’s apparent tumour suppressor role in tumourigenesis, however no study has investigated the nature of ZBTB4’s ability to interact with both the Z4BS and the KBS <em>in vivo</em>, and how this may expand ZBTB4’s repertoire of potential target genes.</p> <p>Recently Kaiso has been characterized as a transcriptional repressor of the cell cycle regulatory gene <em>cyclin D1</em>, and thus we used <em>cyclin D1 </em>as a model to investigate the nature of ZBTB4’s interaction with the KBS <em>in vivo</em>. The <em>cyclin D1</em> minimal promoter contains two partial Z4BS at the same location as the KBS sites and we found that ZBTB4 binds to the +69 Z4BS/KBS site, but not to the -1067 site. Because the +69 Z4BS/KBS is immediately flanked by a CpG dinucleotide, this interaction may be a methylation-dependent interaction. To determine the consequence of this interaction, we conducted minimal promoter luciferase assays, and observed that ZBTB4 mediates an activation of the -1748-CD1 minimal promoter activity.</p> / Master of Science (MSc)

<p>Renal cell carcinoma (RCC) is the most frequently lethal of urological cancers. It arises in the lining of the proximal convoluted tubule of the kidney and is most common in men ages 50–70. Often, partial or radical nephrectomy is needed to effectively treat the disease, leaving patients with reduced kidney function. RCC frequently displays significant radiation resistance, limiting the usefulness of traditional radiation therapy which might spare patients’ normal tissue. The enzyme carbonic anhydrase 9 (CA9), a product of the hypoxia pathway, is found upregulated in the majority of RCC, particularly the clear cell type. It catalyses the dissolution of carbon dioxide into water as bicarbonate and has been linked to increased invasion and migration in RCC tumour cells. The radiation resistance of two RCC cell lines 786-O (human CCRCC) and RAG (murine renal adenocarcinoma) was investigated by the clonogenic assay in the presence of a CA9 inhibitor or silencing RNA. The interference with CA9 by either of these methods significantly sensitizes 786-O cells to the effects of ionizing radiation <em>in vitro</em>. Moreover, fractionation of the dose delivered can increase this sensitization effect. It is hoped that current targeting of CA9 can make radiation therapy a more feasible option in the treatment of RCC.</p> / Master of Science (MSc)

Tumor metabolism is a highly dysregulated process that is identified as a unique target for therapy. Current philosophy proposes that tumor metabolism is a plastic and flexible process which sustains proliferative and survival advantages. Tumors employ an anaerobic glycolytic pathway resulting in the overproduction of lactate. Additional thinking suggests that the conversion of pyruvate to lactate regenerates the NAD+ pool in the cell, maintaining a sustainable oxidative environment. Regardless of the reasons for lactate overproduction, its excretion and build up in the microenvironment results in acidic tumor microenvironments. Tumor acidosis has been measured with several different methods, but consistently averages from pH 6.6 to 7.0. Tumor acidity can thus be measured as a biomarker for tumor metabolism. This work examines the commonly explored energy pathways available to the cancer cell and a non-invasive MRI method to measure the efficacy of the tumor metabolism targeting agent. Appendix A is an introduction to tumor metabolism pathways and the large list of candidate therapies in interfering with energy production. Glucose, fatty acid, and glutamine metabolisms are all discussed along with PI3K/AKT/mTOR and HIF growth signals and ion transport. Magnetic resonance imaging and positron emission tomography are examined as imaging methods for non-invasively interrogating tumor acidosis. Appendix B presents the findings in a study where tumor metabolism was targeted with an mTOR inhibitor, where tumor growth rate was initially decreased and accompanied by an early, acute increase in tumor extracellular pH with acidoCEST MRI. Chapter 2 discusses the combination of a lactate dehydrogenase inhibitor in conjunction with doxorubicin in a breast cancer model. Tumor extracellular pH was shown to increase when measured with acidoCEST MRI, and an increase in cell death was measured. Chapter 4 discusses the studies and experimental designs that can be done in the near future.