Single minded 2 (Sim2) is a member of the basic helix-loop-helix Per-ARNT-Sim (Period-Arylhydrocarbon Nuclear Translocator-Single minded) family. Human SIM2 is involved in the etiology of the Down’s phenotype. In addition to the physical and mental deficiencies associated with DS, it has become apparent that women with DS are 10-25 times less likely to develop breast cancer in comparison to age-matched normal populations. Such significant effects on breast cancer susceptibility are thought to result from gene dosage effects of one or more tumor suppressor genes on chromosome 21. Here we report the identification and transcriptional characterization of mouse Sim2s, a splice variant of Sim2, which is missing the carboxyl Pro/Ala-rich repressive domain. Similar to full-length Sim2, Sim2s interacts with ARNT and to a lesser extent, ARNT2. The effects of Sim2s on transcriptional regulation through hypoxia-, dioxin- and central midline response elements are different than that of full length Sim2. Specifically, Sim2s exerts a less repressive effect on hypoxia-induced gene expression than full length Sim2, but is just as effective as Sim2 at repressing TCDD-induced gene expression from a dioxin response element. Interestingly, Sim2s binds to and activates expression from a central midline response element-controlled reporter through an ARNT transactivation domain-dependent mechanism. Forced expression of SIM2s in MDA-MB-435 breast cancer cells significantly inhibited proliferation, reduced anchorage-independent growth, and decreased invasive potential. SIM2s directly decreased expression of matrix metalloprotease-3, a known mediator of breast cancer metastasis. In addition, loss of Sim2 in the mouse mammary gland increased ductal branching, accelerated lobuloalveolar-like precocious hyperplasia, and decreased cell apoptosis, suggesting that SIM2s is a mammary tumor suppressor. Sim2-/- mammary glands lose E-cadherin expression, suggesting that Sim2s plays a role in regulating E-cadherin/beta-catenin signaling. Loss of Sim2 in the mammary glands also resulted in dramatically increased MMP3 expression. The mechanism of SIM2smediated repression of MMP3 was found to be due to its ability to inhibit AP-1 binding to the MMP3 promoter. These results suggest that SIM2s contributes to the breast cancer protective effects observed in DS individuals.

Abdominal Aortic Aneurysm (AAA) is a complex disease that leads to a localized dilation of the infrarenal aorta that develops over years. Longitudinal information in humans has been difficult to obtain for this disease, therefore mouse models have become increasingly used to study the development of AAAs. The objective of this study was to determine any changes that occur in the biomechanical response and fiber microstructure in the apolipoprotein E difficient (ApoE-/-) angiotensin II (AngII) infused mouse model of aneurysm during disease progression, as well as determine some of the underlying changes in biochemistry, and demonstrate a novel method of reducing any pathogenic protease activity. Using a Microbiaxial Opto-Mechanical Device (MOD), ex vivo studies included adult aortas of ApoE-/- AngII infused mice excised and tested for mechanical response simultaneously imaged using two-photon microscopy to assess the microstructure at multiple time points. In vitro and ex vivo studies have shown changes in protease concentrations with the use of FRET based proteolytic beacons able to provide a non-destructive method to quantify protease activity measured against mechanical and microstructural changes. In vitro studies have demonstrated protease activity can be reduced using a molecule providing a positive feedback mechanism for protease inhibition and possibly provide a reduction in aneurysm progression.

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a metalloproteinase which becomes upregulated in prostate cancer and has been implicated in processes of prostate cancer metastasis. Here, we show that MT1-MMP is minimally expressed in nonmalignant primary prostate cells, moderately expressed in DU-145 cells, and highly expressed in invasive PC-3 and PC-3N cells. Using MT1-MMP promoter reporters and mobility shift assays, we show that Sp1 regulates MT1-MMP expression in DU-145, PC-3, and PC-3N cells and in PC3-N cells using chromatin immunoprecipitation analysis and silencing RNA. Investigation of signaling pathways in these cells showed that DU-145 cells express constitutively phosphorylated extracellular stress-regulated kinase (ERK), whereas PC-3 and PC-3N cells express constitutively phosphorylated AKT/PKB and c-Jun NH2 terminal kinase (JNK). We show that MT1-MMP and Sp1 levels are decreased in PC-3 and PC-3N cells when PI-3K and JNK are inhibited, and that MT1-MMP levels are decreased in DU-145 cells when MEK is inhibited. Transient transfection of PC-3 and PC-3N cells with a dominant-negative JNK or p85, and DU-145 cells with a dominant negative ERK, reduced MT1-MMP promoter activity. We also identified the insulin-like growth factor (IGF-1R) as an upstream regulatory component of MT1-MMP in PC-3N and LNCaP cells, which express high and low levels of the enzyme, respectively. Treatment of PC-3N cells with an IGF-1R specific inhibitor decreased MT1-MMP promoter activity, RNA and protein levels. Additionally, treatment of LNCaP cells with a synthetic androgen to increase IGF-1R levels and subsequent treatment with IGF-I increased MT1-MMP promoter activity, RNA and protein levels. Analysis of MT1-MMP and IGF-1R expression in human prostate cancer tissues demonstrated that MT1-MMP expression was high in the apical cytoplasmic regions of PIN and prostate cancer and less intense in the basalateral cytoplasmic membrane regions of benign glands. IGF-1R was expressed in normal glands and highly expressed in prostate cancer. In conclusion, we have identified several novel mechanisms regulating MT1-MMP expression in prostate cancer cell lines as well as differential localization of the enzyme in human prostate cancer tissues. These results provide insight into the complex mechanisms of prostate cancer metastasis and may be useful for developing future diagnostic procedures or therapies.

Proteases of the plasminogen activator (PA) and the matrix metalloproteinase (MMP) enzyme systems are expressed in the ovulatory follicle and in the developing corpus luteum (CL). However, the functional role of these extracellular degrading protease systems in the ovulatory and CL development processes remains elusive. The first aim of this thesis was to develop a mouse model to study gonadotropin-induced CL formation. The second aim was to study the involvement of the PA and the MMP systems in gonadotropin-induced ovulation, and in CL formation and function. A mouse model for gonadotropin-induced CL formation was developed in order to control the timing of CL formation. In this model, immature mice were induced to ovulate by administrating gonadotropins and the endogenous prolactin surges were mimicked by administration of prolactin twice daily from day 2 of CL development. We observed that steroidogenic acute regulatory protein (StAR) mRNA was highly expressed at days 3 and day 6 of CL development and the levels remained high until late stages of CL regression. Since mice lacking plasminogen (plg-/-) only have a 14% reduction of ovulation efficiency, our hypothesis was that the MMP system could compensate for the loss of plasminogen. When administrating the MMP-inhibitor galardin to gonadotropin-primed ovulating mice, we found that wild-type mice (plg+/+ and C67BL/J6) and heterozygous mice (plg+/-) had an 18-20% reduction in ovulation efficiency as compared to untreated mice. Two models for CL formation, the adult pseudopregnant (psp) mouse model and a model whereby immature gonadotropin-primed mice were treated with prolactin, were used to study the formation and function of the CL in plg-/- mice treated with galardin. At day 3 of CL development, we found no alterations other than a slightly lower number of CL in plg-/- mice. This is most likely a secondary effect of the lower ovulation efficiency found in these mice. On the other hand, we found a 54% reduction in serum progesterone levels in plg-/- mice and a 37% reduction in the plg+/- mice as compared to wild type mice. At day 6 of CL development we saw a 45 % reduction of serum progesterone level in the plg-/- mice and a 22 % reduction in the plg+/- mice. A similar trend was observed at day 3 of CL development in immature gonadotropinprimed mice treated with prolactin. Galardin treatment did not alter the results significantly and the CLs were healthy and viable in these mice. In conclusion, our data suggest that both plasminogen and MMPs, alone or in combination, are dispensable for ovulation and for the formation of a viable CL under the conditions used in this study. The reduced serum progesterone levels observed in the plg-/- mice did not appear to be a result of defective CL formation. Instead, plasmin may have a novel role in the maintenance of luteal function. StAR expression may also be a good marker for CL development and regression in mice.

Transplantation is the optimal form of therapy for patients with end-stage liver disease; however, the use of organs with hepatic steatosis is often associated with increased risks for poor function and graft loss. In addition, ischemia reperfusion (IR) injury leads to cellular damage that can culminate in functional impairment and loss of graft. Furthermore, IR injury is aggravated by pre-existing steatosis and may involve additional mechanisms and mediators of cellular damage. Current models to study IR in vitro are not well defined and may overlook periods of injury that are involved in transplantation. In this thesis, I present an in vitro model for IR injury that includes multiple phases of injury and leads to the upregulation of heme oxygenase-1 (HO-1), and possibly enhances the expression of matrix metalloproteinase-9 (MMP-9). As graft HO-1 expression correlates positively with reduced injury, but MMP-9 expression is associated with increased injury, I therefore examined the utility of in vitro gene therapies to affect the expression of these proteins. We conclude that the in vitro model of ischemia and reperfusion is a promising tool to study the cellular response to IR and may provide a platform for the development of future therapies which could have clinical applications.

In Canada, colorectal cancer is the second leading cause of cancer death for both men and women. There are many different factors that contribute to the progression and spread of the disease. However, increasing evidence now suggests that the tumor microenvironment plays a paramount role in these processes. CD26 is a multifunctional, cell-surface glycoprotein that has intrinsic enzyme activity, binds adenosine deaminase and interacts with the extracellular matrix. Through its various functions it serves to constrain cancer progression. For example, it is known to cleave CXCL12, the ligand for CXCR4. The CXCL12:CXCR4 axis is normally involved in cancer metastasis by promoting cancer cell migration, invasion and proliferation. Down-regulation of CD26 is observed in certain cancers - this has been shown in vitro to occur in response to certain soluble mediators. The first part of this study looked at the effects of glucose and its metabolic product lactate on CD26 expression in colorectal carcinoma cells. Our study showed that CD26 expression is lower in cancer cells that are grown in low-glucose, high-lactate conditions, which replicates the situation within a tumor. The second part of this study examined the effect of adenosine, a purine nucleoside, on colorectal carcinoma cells and supportive stromal cells - cancer-associated HS675.T fibroblasts (CAFs) and Met-5a mesothelial cells. Adenosine increased the proliferation of CAFs and increased CXCL12 mRNA in both stromal cell lines. It also increased MMP-13 mRNA in stromal cells as well as colorectal cancer cells, suggesting that adenosine may promote progression and metastasis through various mechanisms. The last section focused on the ability of cellular products and 3-dimensional tissue topology to coordinate and affect the behaviour of the different cell populations. Here we show that secretory products from colorectal cancer cells promote CAF proliferation but inhibit mesothelial cell proliferation, and are also able to modulate MMP-13 expression. Finally, certain responses are enhanced in multicellular spheroids. In conclusion, the tumor microenvironment represents a major consideration in the treatment of solid tumors. Our data suggest that various soluble mediators, such as adenosine, may have therapeutic implications in cancer treatment and might represent novel targets for future research.

Cardiovascular disease is one of the main causes of mortality and one of the significant burdens to society. Major cardiovascular diseases such as acute myocardial infarction (heart attack), heart failure and cardiac arrhythmia often result in the development of ischemia/reperfusion (I/R) injury. Untreated I/R injury is known to cause cardiac contractile dysfunction. It is established that matrix metalloproteinase-2 (MMP-2) is activated and degrades contractile proteins during I/R, and many other factors including metabolic enzymes, kinases and structural proteins are affected by I/R. However, the molecular mechanisms responsible for these changes are unclear. Since MMP-2 is known to its broad spectrum of action, I hypothesize that, in addition to contractile proteins, proteins related to regulation of energy metabolism are MMP-2 targets during I/R, and protein kinase such as myosin light chain kinase (MLCK) is also involved in this process. The use of proteomics in studying heart injury triggered by I/R will reveal new potential targets for pharmacological protection of heart from I/R induced contractile dysfunction. In addition, selective inhibition of MMP-2 using MMP-2 siRNA protects the heart from I/R injury. In this study, we investigated the protein modulation during I/R using proteomic approach. In order to study the effect of protein kinases (MLCK) and MMP-2, their selective inhibitors were used to inhibit those factors and evaluate the changes in energy metabolic proteins during I/R. Proteomic analysis revealed that six proteins are involved in energy metabolism: ATP synthase β subunit, cytochrome b-c1 complex subunit 1, 24-kDa mitochondrial NADH dehydrogenase, NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, cytochrome c oxidase subunit, and succinyl-CoA ligase subunit, resulting in decreased levels in I/R hearts. The data suggests that energy metabolic proteins, especially the metabolic enzymes involved in the electron transport chain in the mitochondria may contribute to I/R injury. In addition, our data provides evidence that the right and left ventricles of the heart respond differently to I/R injury, in terms of the regulation of contractile proteins and energy metabolic enzymes. Studies using MLCK inhibitor, ML-7, and MMP-2 inhibitor, MMP-2 siRNA to investigate the effect of myosin light chain kinase (MLCK) and MMP-2 in energy metabolic proteins have shown that succinyl-CoA ligase and ATP synthase are affected by MLCK and MMP-2 respectively. These results demonstrate that the effect of inhibition of the MLCK and MMP-2 involves optimization of energy metabolism in I/R injury, likely resulting in increased energy production. Hence, the observed proteins increase in cardiac recovery after I/R. Also, inhibition of MLCK and MMP-2 by ML-7 and MMP-2 respectively shows cardio protective effect during I/R. In summary, this study provides a novel pathogenesis in the development of I/R-induced cardiac contractile dysfunction. Moreover, we suggest a new therapeutic approach whereby using MMP-2 siRNA can be a promising gene therapy in the development of new preventive or treatment strategies against I/R injury.

Ischemic stroke is characterized by a disruption of blood supply to a part of the brain tissue, which leads to a focal ischemic infarct. The expression and activity of MMP-9 is increased in ischemic stroke and is considered to be one of the main factors responsible for damages to the cerebral vasculature, resulting in compromised blood-brain barrier (BBB) integrity. However, the regulatory mechanisms of MMP-9 expression and activity are not well established in ischemic stroke. Since hypoxia/ischemia and reperfusion generates reactive oxygen species (ROS), I hypothesize that ROS is one of factors involved in up-regulation of MMP-9 expression in brain cells and ROS-mediated effect may occur via MAPK signaling pathway. My study has provided the evidence that ROS is responsible for an increase in MMP-9 expression in astrocytes mediated via MAPK-AP1 signaling pathway. Preliminary studies with an in vitro model of the BBB suggest that inhibition of MMP-9 is a critical component of reducing ROS-induced BBB permeability.

Made available in DSpace on 2014-12-17T15:32:28Z (GMT). No. of bitstreams: 1 Tese_GeorgeJFN.pdf: 2720479 bytes, checksum: 6d72db8b12a0e5f743d69a67430d66b7 (MD5) Previous issue date: 2010-02-18 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Matrix metalloproteinase-7 (MMP-7) and -9 (MMP-9) modulate important functions strictly related to the development, invasion and metastasis of several human cancers among them the squamous cell carcinoma of the tongue (SCCT). However, individual genetic factors such as the functional single nucleotide polymorphisms (SNPs) influence the pattern of protein expression of these MMPs and thus may be related to the variability observed in the clinical behavior of patients with SCCT. In this context, the present cross-sectional study aimed to evaluate the association between the frequency of the functional SNPs MMP-7 -181 A/G and MMP-9 -1562 C/T and the clinical (age, gender and metastasis) and pathological (malignancy histological grading and immunohistochemistry expression) features of SCCT cases. Genotyping of these SNPs were performed by PCR-RFLP on DNA samples from 71 cases of SCCT and 60 individuals without cancer who constitute the control group. Among the results of this research, it was observed that the frequency of the polymorphic alleles MMP-7 -181 G and MMP-9 -1562 T in SCCT patients was 28% and 12%, respectively, and the frequency of the heterozygotes A/G (PR = 2.00; p < 0.001) and C/T (PR = 1.54; p = 0.014) were significantly higher in the patient group than in the controls. The prevalence of patients carrying the combination of SNPs studied was significantly associated with SCCT cases (PR = 2.00; p = 0.011) and metastasis (PR = 2.00; p < 0.001). Furthermore, with the frequency of SNPs analyzed, the age, gender, histological grading and immunoreactivity of MMP-7 and MMP-9 formed clinical and pathological parameters relevant to the identification of population subgroups more related to the development of SCCT and metastasis. Based on these results, it is suggested that the protein expression levels of MMP-7 and -9 substantially influence the balance between their pro- and anticancer biological functions and hence the clinicopathological profile of the squamous cell carcinoma of the tongue / As metaloproteinases da matriz extracelular-7 (MMP-7) e -9 (MMP-9) modulam importantes fun??es relacionadas ao desenvolvimento, invas?o e met?stase de diversos c?nceres humanos, dentre os quais o carcinoma epiderm?ide de l?ngua (CEL). Entretanto, fatores gen?ticos individuais, tais como polimorfismos de nucleot?deo ?nico (SNPs) funcionais, influenciam no padr?o de express?o proteica dessas MMPs, podendo estar relacionados ? variabilidade no comportamento cl?nico tumoral observado em pacientes com CEL. Neste contexto, o presente trabalho objetivou, atrav?s de an?lise em sec??o transversal, estudar a associa??o entre a frequ?ncia dos SNPs funcionais MMP-7 -181 A/G e MMP-9 -1562 C/T e as caracter?sticas cl?nicas (idade, sexo e met?stase) e patol?gicas (grada??o histol?gica e express?o imuno-histoqu?mica) em uma s?rie de casos de CEL. A genotipagem dos referidos SNPs foi executada por PCR-RFLP em amostras de DNA de 71 casos de CEL e de 60 indiv?duos sem c?ncer, que constitu?ram o grupo controle. Dentre os resultados da presente pesquisa, evidenciou-se que a frequ?ncia dos alelos polim?rficos MMP-7 -181 G e MMP-9 -1562 T nos pacientes com CEL foi de 28% e 12%, respectivamente, sendo as frequ?ncias dos heterozigotos A/G (RP = 2.00; p < 0.001) e C/T (RP = 1.54; p = 0.014) significativamente maiores neste grupo de pacientes que no grupo controle. A preval?ncia dos pacientes portadores da combina??o dos SNPs estudados associou-se significativamente aos casos de CEL (RP = 2.00; p = 0.011) e ? met?stase (RP = 2.00; p < 0.001). Ademais, junto ? frequ?ncia dos SNPs analisados, a idade, sexo, grada??o histol?gica e imunoexpress?o da MMP-7 e -9 constitu?ram par?metros clinicopatol?gicos relevantes para a identifica??o de subgrupos populacionais mais predispostos ao desenvolvimento do CEL e met?stase. Frente a estes resultados, sugere-se que os n?veis de express?o da MMP-7 e -9 influenciam consideravelmente no balan?o entre suas fun??es pr? e antineopl?sicas e, consequentemente, no perfil clinicopatol?gico do carcinoma epiderm?ide de l?ngua.

Abstract Bladder cancer when superficial has a good prognosis but it has a high recurrence risk and about 10–15% of the superficial carcinomas will progress into muscle invasive or metastatic type. The most powerful factor for predicting the behavior of bladder carcinoma is the stage of the tumor. Invasion to the lamina propria increases the risk of recurrence and progress to muscle-invasive tumor. Also grade of the tumor and tumor multiplicity associates with high risk for recurrence. New markers are still needed to find those patients who need more and better treatments to avoid the recurrence and progress. The need for new non-invasive markers to diminish the need for frequent cystoscopy in follow-up is also obvious. Gelatinases MMP-2 and MMP-9 are known to associate to tumor invasion and progression. Also their tissue inhibitors TIMP-1 and TIMP-2 take part in these diversified processes and metastasis formation. In the present work the expression and clinical value of gelatinases MMP-2 and MMP-9 and their tissue inhibitors TIMP-1 and TIMP-2 were evaluated in bladder carcinoma. Primary tissue samples of 121 patients were analyzed for expression of MMP-2 and/or MMP-9 using immunohistochemistry. The serum samples of 87 patients who were treated in the Oncology Department of Oulu University Hospital were collected and studied with ELISA. The control group consisted of 44 healthy volunteers. Overexperssion of MMP-2 protein correlated significantly to disease-specific survival and showed an independent prognostic value as a biomarker. High MMP-9 expression instead correlated to favorable overall survival of bladder cancer patients. Circulating proMMP-2, TIMP-2 and MMP-2:TIMP-2 complex levels were lower in cancer patients than in healthy volunteers in control group. High levels of all these three markers correlated with better prognosis in bladder cancer patients.