A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Glioblastoma (GB) is the highest grade and most common form of primary adult brain tumors, characterized by a highly invasive cell population. GB tumors develop treatment resistance and ultimately recur; the median survival is nearly fifteen months and importantly, the invading cell population is attributed with having a decreased sensitivity to therapeutics. Thus, there remains a necessity to identify the genetic and signaling mechanisms that promote tumor spread and therapeutic resistance in order to develop new targeted treatment strategies to combat this rapidly progressive disease. TWEAK-Fn14 ligand-receptor signaling is one mechanism in GB that promotes cell invasiveness and survival, and is dependent upon the activity of multiple Rho GTPases including Rac1. Here, we show that Cdc42 is essential in Fn14-mediated Rac1 activation. We identified two guanine nucleotide exchange factors (GEFs), Ect2 and Trio, involved in the TWEAK-induced activation of Cdc42 and Rac1, respectively, as well as in the subsequent TWEAK-Fn14 directed glioma cell migration and invasion. In addition, we characterized the role of SGEF in promoting Fn14-induced Rac1 activation. SGEF, a RhoG-specific GEF, is overexpressed in GB tumors and promotes TWEAK- Fn14-mediated glioma invasion. Moreover, we characterized the correlation between SGEF expression and TMZ resistance, and defined a role for SGEF in promoting the survival of glioma cells. SGEF mRNA and protein expression are regulated by the TWEAK-Fn14 signaling axis in an NF-B dependent manner and inhibition of SGEF expression sensitizes glioma cells to TMZ treatment. Lastly, gene expression analysis of SGEF depleted GB cells revealed altered expression of a network of DNA repair and survival genes. Thus TWEAK-Fn14 signaling through the GEF-Rho GTPase systems which include the Ect2, Trio, and SGEF activation of Cdc42 and/or Rac1 presents a pathway of attractive drug targets in glioma therapy, and SGEF signaling represents a novel target in the setting of TMZ refractory, invasive GB cells.

In a recent patent, GPR55 emerged as a novel target for cannabinoid modulation, and was found to be activated by a number of synthetic and endogenous cannabinoids, including anandamide, virodhamine, and AM251.  There is mounting evidence to suggest that cannabinoids play a role in tumour progression.  The potential role of GPR55 in cancer, however, remains to be explored. This study used qPCR to determine GPR55, CB₁ and CB₂ expression levels in a number of native breast cancer cell lines, and found that GPR55 was expressed in highly metastatic cells, including MDA-MB-231 and B02-GFP cell lines. Studies into the modulation of GPR55 by its suggested ligands showed that agonist L-α-lysophosphatidylinositol enhanced the migration of metastatic breast cancer cells, and augmented certain behaviours associated with a more aggressive phenotype, including enhancing the ability of these cells to orientate and polarise in response to grooved substratum.  Meanwhile the GPR55 antagonist CBD inhibited the migration and invasion of these cells.  Using over-expression and siRNA knockdown of GPR55, we have implicated this receptor in both the LPI- and CBD-mediated effects on migration/invasion.  Furthermore, we found that LPI significantly stimulated both RhoA activation, and ERK1/2 phosphorylation in hGPR55-expressing HEK293 cells, but not in untransfected HEK293 cells.  These studies highlight an important role for GPR55 as a new biomarker in cancer, and suggest a possible therapeutic role for cannabinoids in the treatment of metastasis.

In this thesis, the theory of poroelasticity, namely the Mixture Theory version -- a homogenized, macroscopic scale approach used to describe fluid flow through a porous medium -- is employed in three separate cases pertaining to a biological phenomenon. The first investigation explores the behavior of interstitial fluid pressure (IFP) in solid tumors. Thus, in Chapter 2, a Mixture Theory based approach is developed to describe the evolution of the IFP from that in a healthy interstitium to the elevated levels in cancerous tumors. Attention is focused on angiogenesis, a tightly regulated process in healthy tissue that provides all necessary nutrients through the creation of new blood vessels. Once this process becomes unruly within a tumor, angiogenesis gives rise to an abnormal vasculature by forming convoluted and leaky blood vessels. Thus, the primary focus of the model is on the capillary filtration coefficient and vascular density as they increase in time, which in turn elevates the tumor IFP. Later, the Mixture Theory model is extended to simulate the effects of vascular normalization, where the cancer therapy not only prunes blood vessels, but reverts the chaotic vasculature to a somewhat normal state, thereby temporarily lowering the tumor IFP. In Chapter 3, the validity of an assumption that was made in order to facilitate the mathematical calculations is investigated. In addition to all of the Mixture Theory assumptions, it is assumed that the pore pressure p is proportional to the tissue dilatation e. This assumption is examined to determine how appropriate and accurate it is, by using a heat type equation without the presence of sources and sinks under the assumption of a spherical geometry. The results obtained under the proportionality of p and e, are compared with the results obtained without this assumption. A substantial difference is found, which suggests that great care must be exercised in assuming the proportionality of p and e. The last application is reported in Chapter 4 and it investigates the pathogenesis of normal pressure hydrocephalus. In a normal brain, cerebrospinal fluid (CSF) is created by the choroid plexus, circulates around the brain and the spinal cord without any impediment, and then is absorbed at various sites. However, normal pressure hydrocephalus occurs when there is an imbalance between the production and absorption of CSF in the brain that causes the impaired clearance of CSF and the enlargement of ventricles; however, the ventricular pressure in this case is frequently measured to be normal. Thus, a mathematical model using Mixture Theory is formulated to analyze a possible explanation of this brain condition. Levine (1999) proposed the hypothesis that CSF seeps from the ventricular space into the brain parenchyma and is efficiently absorbed in the bloodstream. To test this hypothesis, Levine used the consolidation theory version of poroelasticity theory, with the addition of Starling's law to account for the absorption of CSF in the brain parenchyma at steady state. However, the Mixture Theory model does not agree with the results obtained by Levine (1999) which leads one to conclude that the pathogenesis of normal pressure hydrocephalus remains unknown. To conclude the thesis, all three applications of Mixture Theory are discussed and the importance and contribution of this work is highlighted. In addition, possible future directions are indicated based on the findings of this thesis.

EGFR (Epidermal Growth Factor Receptor) is a protein that is ubiquitous in the human body. Aberrant activity of EGFR or its isoforms is implicated in a number of cancers, notably brain cancer. An isoform of EGFR, EGFRvIII (EGFR variant III), is particularly relevant to brain cancer since it is only naturally found in brain tumour tissue. However, the presence and activity of EGFRvIII is not well characterized. I hypothesize that the different levels of EGFRvIII expression and its expression relative to wild type EGFR in human brain tumour tissue can be used to diagnose the different stages and progression of disease in the glioblastoma multiforme (GM) type of brain cancer. The work presented in this thesis is an attempt to develop a method for the accurate and absolute quantitation of EGFRvIII from brain tumour tissue. Using iMALDI (immunoMALDI), which combines the high-specificity of MALDI mass spectrometry with antibody immunoaffinity enrichment, I have optimized and developed a highthroughput technique for sensitive, specific and quantitative detection and differentiation of EGFR and EGFRvIII. I have also demonstrated a proof-of-concept by applying this assay to the isolation and detection of these proteins from brain tumour tissue. / Graduate

No description available.

Angiogenesis is the formation of new vessels from existing vasculature and is essential for tumour growth and metastasis. It is controlled by angiogenic factors secreted by the tumour which regulate the matrix remodelling, endothelial cell (EC) proliferation, and capillary differentiation necessary for establishing a blood supply. This thesis has examined angiogenesis in human tumours. Immunohistochemically highlighted vessels in tumours were quantified using different methods to develop a rapid and objective method for measuring tumour angiogenesis. Significant associations between Chalkley counting, microvessel density, vascular area and perimeter were demonstrated; the Chalkley technique gave independent prognostic information and was suitable for a diagnostic service. Studies on the frequency EC of S-phase showed proliferation (labelling index 2.2%) occurs mostly at the tumour margin suggesting the growth factors controlling ECs are different from those regulating tumour cells and that remodelling the existing vasculature might play a more important role than previously recognised. To investigate further ECtumour matrix interactions, the expression of cell adhesion molecules (CAMs) was examined. CAM expression mirrored that of EC proliferation with preferential expression on endothelium at the tumour periphery: expression of CAMs was also present on neoplastic cells. Thus, acquisition of CAMs by tumour cells together with EC phenotypic modulation might promote angiogenesis and metastasis. The angiogenic factor thymidine phosphorylase (TP) was examined in normal tissues and tumours. Although TP was expressed in ECs there was no correlation between expression in normal or neoplastic tissue and vascularity. Nevertheless, TP was elevated in small low grade tumours, in accordance with TP being chemotactic but non-mitogenic for ECs. A monoclonal antibody to flt-4, a candidate angiogenic factor receptor was generated and characterised. In contrast to the in-situ mRNA expression profile, a restricted pattern of protein expression was observed in normal tissues and variable expression in tumours.

Endothelin-1 (ET-1) is a potent vasoactive peptide, cleaved from its inactive precursor, big ET-1, by Endothelial Converting Enzyme-1 (ECE-1). ET-1 normally regulates vascular tone via its two receptors ET-A and ET-B. As ET-1 is found to be over-expressed in many tumour models, it has been implicated as having a role in neovascularisation. / Thesis (PhDBiomedicalScience)--University of South Australia, 2005.

Thesis (Ph. D.)--Washington State University, August 2008. / Includes bibliographical references (p. 88-101).

Thesis (M. Res.(Med.))--University of Hong Kong, 2008. / Includes bibliographical references (p. 68-76)

Thesis (M. Med. Sc.)--University of Hong Kong, 2008. / Includes bibliographical references (p. 52-55)