The Hippo-LATS signaling pathway originally identified in Drosophila is conserved in mammalian systems and serves essential roles in mediating size control as well as tumorigenesis. In humans, the core kinase cassette consisting of adaptor proteins WW45 and MOB1, and Ser/Thr kinases MST1/2 and LATS1/2 signal by phosphorylating and inactivating transcriptional co-activators YAP and TAZ, causing cell growth arrest. As the central kinases within the Hippo-LATS pathway, examining the cellular and molecular phenotypes of LATS1 and LATS2 (LATS) will provide insight into the role of this pathway in tumorigenesis. By simultaneously knocking down both LATS1 and LATS2, genes that were differentially expressed were identified through a whole human genome microarray screen. The multitude of genes identified including CYR61, MYLK, CDKN1A, SLIT2, and TP53INP1 not only provide further evidence for the role of LATS in cell proliferation and apoptosis, but also implicate LATS in novel functions such as cell motility. Loss of LATS1 and/or LATS2 enhances cell migration whereas overexpression of LATS1 dramatically inhibits cell migration in multiple cell lines. The ability of LATS to regulate cell migration occurs through two potential mechanisms. Firstly, LATS functions through its kinase substrates YAP and/or TAZ, or alternatively, LATS1 directly binds actin and inhibits actin polyermization. Thus, through loss of functions studies, we identified a novel role for LATS in regulating cell migration as well as novel mechanisms of LATS function. As an important signaling molecule within the cell, LATS and the Hippo-LATS pathway are tightly regulated. Using clues from the Drosophila pathway, we examined how the previously uncharacterized gene, hEx, functions within this pathway. Importantly, this thesis characterizes hEx as a putative tumor suppressor showing that it can inhibit cell proliferation, sensitize cancer cells to Taxol treatment as well as inhibit tumor growth in nude mice. However, unlike Drosophila expanded, hEx functions independently of the Hippo-LATS pathway, suggesting that the mammalian signaling pathway is more complicated. The research findings from this thesis enhance our knowledge of the Hippo-LATS pathway in tumorigenesis by elucidating new functions and mechanisms of LATS functions as well as by exploring how upstream components function in relation to this pathway. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2011-09-23 10:06:54.687

Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). Antigen induced activation of Th1 cells in the peripheral blood leads to elevated production of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) that have been directly linked to disease induction and neuropathic pain. It was hypothesized that following antigenic induction, cytokines gain access to the spinal cord and participate in direct cellular interaction with dorsal horn neurons. Using an animal model of MS, we show that TNF-α gene and protein expression in the dorsal root ganglia (DRG) and spinal cord tissue is increased in the active group. In addition, our findings show TNF-α mRNA expression in the dorsal root entry point. Therefore, our results support the hypothesis that antigen induced DRG derived TNF-α can transport to the spinal cord via the dorsal roots and is involved in the underlying pathogenesis of MS induced neuropathic pain.

Background: Multiple sclerosis (MS) is a chronic, neurological disease characterized by targeted destruction on central nervous system (CNS) myelin. The autoimmune theory is the most widely accepted explanation of disease pathology. Circulating Th-1 cells become activated by exposure to CNS-specific antigens such as myelin basic protein. The activated Th-1 cells secrete inflammatory cytokines, which are pivotal for inflammatory responses. We hypothesize that enhanced production of inflammatory cytokines triggers cellular events within the dorsal root ganglia (DRG) and/or spinal cord, facilitating the development of neuropathic pain (NPP) in MS. NPP, the second worst disease-induced symptom suffered by patients with MS, is normally regulated by DRG and/or spinal cord. Objective: To determine gene and protein expression levels of tumor necrosis factor-alpha (TNF ) within DRG and/or spinal cord in an animal model of MS. Methods: Experimental autoimmune encephalomyelitis (EAE) was induced in adolescent female Lewis rats. Animals were sacrificed every 3 days post-disease induction. DRG and spinal cords were harvested for protein and gene expression analysis. Results: We show significant increases in TNF expression in the DRG and of EAE animals at peak disease stage, as assessed by clinical symptoms. Conclusion: Antigen-induced production of inflammatory cytokines such as TNF within the DRG identifies a potential noel mechanism for MS-induced NPP.

This thesis investigated the cooperation of the Kras2 oncogene with the tumor suppressor gene Dlc1 in lung tumor development. Dlc1 is a negative regulator of RhoGTPase proteins, which are mainly involved in the regulation of the actin cytoskeleton and cell migration. We hypothesized that loss of Dlc1 expression leads to more aggressive tumors, which should also result in increased incidence of metastasis. All experiments were performed in mice containing a heterozygous oncogenic Kras allele and a heterozygous gene trapped Dlc1 allele (KD) and in mice only carrying the oncogenic Kras allele (K+). Throughout all experiments we have consistently found no significant differences between the two groups in terms of tumor burden (tumor numbers, sizes and areas), metastases or methylation patterns. These results suggest that heterozygous downregulation of Dlc1 is not enough to increase tumor formation and metastasis development in the Kras lung tumors.

Adaptive radiation therapy re-optimizes treatment plans based on updated tumor geometries from magnetic resonance imaging scans. However, the imaging process is costly in labor and equipment. In this study, we develop a mathematical model that describes tumor evolution based on a Markov assumption. We then extend the model to predict tumor evolution with any level of information from a new patient: weekly MRI scans are used to estimate transition probabilities when available, otherwise historical MRI scans are used. In the latter case, patients in the historical data are clustered into two groups, and the model relates the new patient's behavior to the existing two groups. The models are evaluated with 33 cervical cancer patients from Princess Margaret Cancer Centre. The result indicates that our models outperform the constant volume model, which replicates the current clinical practice.

Adaptive radiation therapy re-optimizes treatment plans based on updated tumor geometries from magnetic resonance imaging scans. However, the imaging process is costly in labor and equipment. In this study, we develop a mathematical model that describes tumor evolution based on a Markov assumption. We then extend the model to predict tumor evolution with any level of information from a new patient: weekly MRI scans are used to estimate transition probabilities when available, otherwise historical MRI scans are used. In the latter case, patients in the historical data are clustered into two groups, and the model relates the new patient's behavior to the existing two groups. The models are evaluated with 33 cervical cancer patients from Princess Margaret Cancer Centre. The result indicates that our models outperform the constant volume model, which replicates the current clinical practice.

In dieser Arbeit wurde in einem Maus Tumormodell untersucht, ob durch eine intraoperative Vakzination mit gentechnisch modifizierten autologen Tumorzellen ein antitumoraler Effekt erzielt werden kann. Das Experiment erfolgte mit zwei Tumorzelllinien (B16 Melanom und Lewis Lung Karzinom). Nach Implantation der Tumorzellen in C57/BL 6 Mäuse wurden diese chirurgisch entfernt. Intraoperativ erhielten die Mäuse eine Vakzination. Dazu wurden folgende Impfstoffe verwendet: 1. subletal bestrahlte mIL-12 transfizierte Tumorzellen, 2. subletal bestrahlte pRSC transfizierte Tumorzellen und 3. frostgeschockte Tumorzellen. Die Impfung erfolgte entweder subcutan oder direkt in die Milz. Es wurde die Hypothese aufgestellt, dass eine Injektion in die Milz und eine Modifikation mit IL-12 den besten Effekt erzielt. Eine Kontrollgruppe blieb ohne Vakzin. Beobachtet wurde das Tumorwachstum, der Zeitpunkt bis zum makroskopischen Wiederauftreten eines Tumors, Überlebenszeit und die Metastasierungsrate. Versuchstiere ohne Rezidivtumor erhielten erneut einen Tumor. Es erfolgte eine erneute Evaluation des Tumorwachstums, des Zeitpunktes bis zum makroskopischen Wiederauftreten eines Tumors, der Überlebenszeit und der Metastasierungsrate. In beiden Tumorzelllinien profitierten alle Therapiegruppen nach Tumorresektion und Vakzination bezüglich Tumorrezidivrate, Zeit bis zum makroskopischenWiederauftreten des Tumors, Überlebenszeit, Metastasierungsrate und Tumorwachstumsgeschwindigkeit gegenüber der Kontrollgruppe. Vereinzelt konnten signifikante Vorteile für die direkt in die Milz applizierte Vakzine bezüglich der Tumorwachstumsgeschwindigkeit aufgezeigt werden. Weiterhin ergab sich eine geringere Tumorrezidivrate, wenn IL-12 modifizierte autologe Tumorzellen nach R0 Resektion direkt in die Milz appliziert wurden. Auch nach Tumorreimplantation konnte bezüglich Überlebenszeit und Tumorwachstumsgeschwindigkeit ein Vorteil für alle Therapiegruppen gegenüber der Kontrollgruppe herausgearbeitet werden. Nach Impfung in die Milz zeigte sich tendenziell eine geringere Metastasierungsrate. Intraoperative autologe Tumorzellvakzinationen konnten im Tiermodell in einem adjuvanten Setting einen antitumoralen Effekt auslösen. Möglicherweise kann diese Art der Impfung eine zusätzlich hilfreiche Behandlungsform zu den bisherigen adjuvanten Chemotherapeutika werden.

The study was conducted to develop a technique that measures the amount of gadolinium based contrast agent accumulated in a head tumour by x-ray fluorescence, while a patient is exposed to neutrons or during external beam radiotherapy planning. In this research, measurements of the gadolinium concentration in a vessel simulating a brain tumour located inside a head phantom, by the x-ray fluorescence method were taken, where the Magnevist contrast medium which has gadolinium atom, in the tumour vessel, was excited by a 36 GBq (0.97 Ci) 241Am source that emits gamma rays of 59.54 keV, in 35.7 % of it’s decays, resulting the emission of characteristic fluorescence of gadolinium at 42.98 keV that appeared in the X-ray fluorescence spectrum. A Cadmium Telluride (CdTe) detector was used to evaluate and make an analysis of the gadolinium concentration. Determinations of the gadolinium content were obtained directly from the detector measurements of XRF from gadolinium in the exposed tumour vessel. The intensity measured by the detector was proportional to the gadolinium concentration in the tumour vessel. These concentrations of gadolinium were evaluated for dose assessment. The positioning of the head phantom was selected to be in the lateral and vertex positions for different sizes of tumour vessels. Spherical tumour vessels of 1.0, 2.0, 3.0 cm and an oval tumour vessel of 2.0 cm diameter and 4.0 cm length, containing the gadolinium agent, contained concentration between 5.62 to 78.63 mg/ml. They were placed at different depths inside a head phantom at different positions in front of the detector and the source for the measurements. These depths ranged from 0.5 cm to 5.5 cm between the center of the tumour and interior wall of the head phantom surface. The total number of measurements in all four sizes of the tumour vessel was 478; 78 examinations of a 1.0 cm spherical tumour vessel, 110 examinations of a 2.0 cm spherical tumour vessel, 150 examinations of a 3.0 cm spherical tumour vessel and 140 examinations of a 2.0 x 4.0 cm ellipsoid tumour vessel. To measure the size and the shape of the tumour by the alternative radiographic method, a general x-ray machine with radiograph film was used. Based on that, the appropriate shape of concentration could be selected for therapy. The differences of optical density in the x-ray films showed that the noise was increased with low concentration of the Gd. Because radiographic film may be subjected to different chemical processes where the darkness will be affected, these measurements would be very hard to be quantitative. Accordingly it is difficult to use the film for Gd concentrations. The obtained data show that the method works very well for such measurements.

This study investigated the interaction between polychlorinated biphenyls (PCBs) and selenium to explain the mechanism involved that could affect selenium metabolism and its anti-cancer property. PCBs congeners and mixtures were previously found to reduce hepatic Se and Se-dependent glutathione peroxidase activity. I hypothesized that certain PCB congeners affect selenium metabolism in the rat liver resulting in diminished antioxidant capacity of selenoproteins, which could alter the ability of Se to protect against PCBs induced tumor promotion. In the first study, the influence of 3,3,4,4-tetrachlorobiphenyl (PCB 77) on hepatic Se and glutathione peroxidase (GPx1) activity as well as cytochrome P450 1A1 induction was examined by employing a time-course study, which showed that PCB 77 significantly reduced the hepatic selenium level and GPx1 activity and that this effect was influenced by gender. The next study explored how PCB 77 could deplete hepatic selenium by determining selenium concentrations in different tissues, feces and urine. This study demonstrated that PCB-77 decreased hepatic Se by increased excretion of Se in urine but not in feces. Unlike glutathione peroxidase, thioredoxin reductase activity was not affected by PCB 77. The third study investigated the effect of selenium supplementation on the tumor promoting activity of PCB 77 and 2,2,4,4,5,5-hexaclorobiphenyl (PCB 153) using a 2-stage carcinogenesis model. Se supplementation did not diminish the induction of altered hepatic foci by coplanar PCB 77 or ortho-substituted PCB 153. Instead of protection, the number of foci per cubic centimeter and per liver among the PCB-77 treated rats was increased as the selenium dietary level increased. PCB 153 did not show the same selenium dose-response effect; nevertheless, selenium supplementation did not confer protection against foci development. On the other hand, supranutritional selenium reduced the mean focal volume. Supranutritional selenium or PCBs did not affect cell proliferation or thioredoxin reductase activity. Lastly, the use of the Zeeman graphite furnace atomic absorption spectrometry (GFAAS) method and closed microwave digestion technique for selenium determination of biological samples was compared with the neutron activation analysis and fluorometry methods. I found that GFAAS was not as reliable as the other methods.

The development of prostate tumors has been linked to co-morbid diabetes mellitus (DM) in several studies, potentially through the stimulation of insulin-like growth factor receptor (IGFR). This study evaluates the effect of anti-diabetic medication use on the development of high grade tumors and time to tumor progression compared to non-diabetics. This retrospective, nested case control study identified patients with prostate cancer (PCa) from the Kentucky Medicaid Database. Cases were diagnosed with PCa and DM and using at least one of the following antidiabetic medications; sulfonylureas, insulin, metformin or TZDs. Cases were further stratified on their insulin exposure resulting from therapy. Controls were those with PCa without DM or any anti-diabetic medications. No statistically significant effects on insulin exposure was found on tumor grade and time to progression. Trends identified that use of metformin or TZDs potentially decreased the odds of high-grade tumors and decreased the risk of progression, while sulfonylureas and high-dose insulin may increase the odds of high-grade tumors and increase the risk of progression compared to non-diabetics. Future studies should be conducted to further evaluate the effects of anti-diabetic medications on tumor grade and time to prostate cancer progression.