Integrin α6β4 is upregulated in pancreatic carcinoma, where signaling promotes metastatic properties, in part by altering the transcriptome. Such alterations can be accomplished through DNA demethylation of specific promoters, as seen with the pro-metastatic gene S100A4. I found that signaling from integrin α6β4 dramatically upregulates expression of amphiregulin (AREG) and epiregulin (EREG), ligands for the epidermal growth factor receptor (EGFR), and that these ligands promote pancreatic carcinoma invasion. To determine if AREG and EREG are regulated by DNA methylation, pancreatic cancer cells with low AREG and EREG expression were treated with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-Aza-CdR), resulting in stable overexpression of AREG and EREG, and this induction required signaling from integrin α6β4. Similarly, treatment of cells with high integrin α6β4 with the methyl donor S-adenosylmethionine inhibited gene expression of AREG and EREG. Whole genome bisulfite sequencing on pancreatic cancer cells reveled hypomethylation of the promoter regions of AREG and EREG when integrin α6β4 is high, and these regions correspond to H3K27Ac, indicative of enhancer location. Interestingly, I also observed genome-wide DNA demethylation, and a large proportion of altered CpGs correspond to potential enhancers. It is currently accepted that active DNA demethylation occurs via DNA repair. I tested this hypothesis by treating cells with Gemcitabine, which inhibits multiple components of DNA repair, including DNA demethylation mediated by GADD45A. Gemcitabine treatment resulted in marked reduction in AREG and EREG expression. To further test the involvement of GADD45A, I used RNAi-mediated knockdown or cDNA overexpression to alter GADD45A levels. In both instances, AREG and EREG expression positively correlated with GADD45A, particularly when integrin α6β4 is high, indicating that GADD45A is a rate-limiting step in AREG and EREG overexpression. Similarly, using stable shRNA, I show that Thymine DNA Glycosylase (TDG), and TET1 known modulators of DNA demethylation, are required for AREG and EREG expression in integrin α6β4 high cells, and nuclear localization of TDG is much higher in cells with high integrin α6β4. Using a specific inhibitor I found that AREG and EREG expression is dependent on Parp-1. Finally, I determined that integrin α6β4 signaling enhances cells ability to respond to and survive in the presence of DNA damage, and that active DNA repair is required for integrin α6β4 mediated DNA demethylation. Taken together, these data indicate that DNA repair is required to maintain overexpression of AREG and EREG in response to signaling from integrin α6β4 and that integrin α6β4 promotes this overexpression by enhancing DNA repair.

Die Entwicklung von Fernmetastasen ist die Haupttodesursache bei Tumorerkrankten und der entscheidende klinisch relevante Schritt während der Tumorprogression. Die Seed and Soil Theorie von Stephen Paget besagt, dass verschiedene Tumorzellen spezifische Zielorgane während der Metatasierung bevorzugen. Während das häufigste Target der kolorektalen Karzinome die Leber ist, hat der triple-negative molekulare Subtyp von Brustkrebs die Neigung, in das Gehirn zu metastasieren. Interessanterweise spielen sowohl deregulierte EGFR (Epithelial growth factor receptor) als auch WNT Signalwege in diesen beiden Entitäten eine entscheidende Rolle. Das Ziel der Arbeit ist, die Rolle der beiden Signalwege in soliden Tumorzelllinien in Bezug auf die Tumorprogression und Kolonisation zu untersuchen. Im Rahmen der molekularen Charakterisierung der Zelllinien zeigten sich die Mammakarzinomzelllinie 410.4 und die kolorektale Tumorzelllinie CMT-93 als passende Modellsysteme für unsere Fragestellung. Anschließend wurden der EGFR und der WNT Signalweg in diesen Zellen im Sinne von gain of function und loss of function moduliert und die Auswirkungen auf Aspekte der Tumorprogression analysiert. In CMT-93 Zellen wurde ein EGFR Knockdown etabliert. Während der Knockdown keinen Einfluss auf die Proliferation hat, vermindert er die Invasion der Zellen. Somit konnte dem effizienten Knockdown eine funktionelle Wirksamkeit zugeschrieben werden. Eine EGFR Überexpression konnte sowohl in 410.4 als auch in CMT-93 Zellen etabliert werden. Die Analyse der jeweiligen Signalkaskadenweiterleitung ergab zwar Änderungen und somit eine funktionelle Relevanz, dies blieb jedoch ohne Auswirkungen auf das Invasionspotential der Zelllinien. Ein Knockdown von β-Catenin konnte in 410.4 zwar etabliert werden, blieb jedoch ohne funktionelle Auswirkungen. Eine stabile Überexpression von β-Catenin war nicht erfolgreich, da dies offenbar mit der Viabilität der Zellen interferierte. Die Relevanz des β-Catenin-abhängigen WNT Signalwegs in den beiden gewählten Zelllinien konnte somit nicht abschließend geklärt werden. Des Weiteren wurde die Bedeutung des nicht-kanonischen WNT Signalwegs via ROR2 und WNT11 untersucht. Dabei ergab sich, dass die Überexpression von WNT11 und ROR2 in 410.4 Zellen deren Invasion durch einen RHOA-abhängigen Mechnismus steigert und einen Einfluss auf den PI3K Signalweg hat. Es ist anzunehmen, dass WNT11 als downstream Target über ROR2 induziert wird und über einen positiven Feedback-Loop via ROR2 eine autokrine Stimulation ausübt.

The introduction of tyrosine kinase inhibitors (TKI) targeting the epidermal growth factor receptor (EGFR) inhibitors to the clinic has resulted in an improvement in the treatment of non small cell lung cancer (NSCLC). However, many patients treated with EGFR TKIs do not respond to therapy. The burden of failed treatment is largely placed on the healthcare field, limiting the effectiveness of EGFR TKIs. Furthermore, responses are hindered by the emergence of resistance. Thus, two questions must be addressed to achieve maximum benefit of EGFR inhibitors: How can patients who will benefit from EGFR TKIs be selected a priori? How can patients who respond achieve maximal benefit? To answer these questions, two hypotheses were formed. First, the EGFR-dependent phenotype, which is displayed by the tumors cells of those patients who respond clinically to EGFR TKIs, can be captured by genomic profiling of NSCLC cell lines stratified by sensitivity to EGFR TKIs. This gene signature may be used to predict the outcome of EGFR TKI therapy in unknown samples. Secondly, the predictive signature of response to EGFR TKI could provide insights into the underlying biology of the phenotype of EGFR-dependency. This information could be exploited to identify inhibitors which could be combined with EGFR inhibitors to elicit a greater effect, thereby minimizing resistance. The work herein describes the testing of these hypotheses. Pharmacogenomics was utilized to define a signature of EGFR-dependency which effectively predicted response to EGFR TKI in vitro and in vivo. Furthermore, the signature was analyzed by bioinformatic approaches to identify the RAS/MAPK pathway as a candidate target in EGFR-dependent NSCLC. The RAS/MAPK pathway regulates expression and activation of EGF-like ligands. Furthermore, the RAS/MAPK pathway modulates EGFR stability in the EGFR-dependent phenotype. Further biochemical analyses demonstrated that the RAS/MAPK pathway mediates proliferation and survival of EGFR-dependent NSCLC cells. Finally, combinatorial treatment of EGFR-dependent NSCLC cell lines with small molecules targeting EGFR and the RAS/MAPK pathway yielded cytotoxic synergy. Thus, we have used pharmacogenomics methods to potentially improve NSCLC treatment by developing a method of predicting response and identifying an additional target to combine with EGFR TKIs to maximize responses.

Les ultraviolets A (UVA) sont carcinogènes et produisent des espèces réactives de l'oxygène (ERO). Le récepteur à la mélanocortine de type 1 (MC1R) est un récepteur couplé aux protéines G (RCPG) qui est impliqué dans la mélanogénèse et dans l'inflammation cutanée. Certains variants du gène sont associés à un risque accru de mélanomes et de carcinomes cutanés. Le MC1R est exprimé surtout dans les mélanocytes mais son expression peut être induite par les UV in vitro dans les kératinocytes et in vivo dans la peau. Le récepteur MC1R est activé par l'α-MSH. L'objectif de ce travail de thèse a été d'étudier les effets du récepteur MC1R sur le stress oxydatif induit par les UVA dans des lignées kératinocytaires humaines HaCaT exprimant le récepteur MC1R ou son variant non fonctionnel Arg151Cys. Nous avons montré que la production d'ERO intracellulaire induite par les UVA est fortement inhibée dans les cellules HaCaT-MC1R et que cette inhibition est renforcée en présence d'α-MSH. L'inhibition du stress oxydatif induit par les UVA dans les cellules transfectées par le MC1R est en partie dépendante de la phosphorylation de la sous-unité activatrice, NoxA1 de la NADPH oxydase. Le traitement des cellules HaCaT-MC1R par un inhibiteur du récepteur au facteur de croissance épidermique (EGFR) restaure l'habilité de ces cellules à induire un stress oxydatif après irradiation UVA. Ces résultats montrent que l'activité constitutive du récepteur MC1R dans des kératinocytes pourrait inhiber le stress oxydatif induit par les UVA via des mécanismes dépendants de l'AMPc et de l'EGFR.

Feline oral squamous cell carcinomas (FOSCCs) are locally aggressive tumours and a common cause of mortality and morbidity. Current treatment options are rarely successful and animals are frequently euthanised upon diagnosis due to their grave prognosis. Epidermal Growth Factor Receptor (EGFR) is a tyrosine kinase receptor which is frequently dysregulated in SCC of the head and neck (HNSCC) in man. Recent advances in human medicine have identified EGFR as a therapeutic target in HNSCC. In this study the role of EGFR in FOSCC was investigated. Sixty seven biopsy samples were immunohistochemically labelled for EGFR and Ki67, a proliferation marker. The tyrosine kinase region of feline EGFR was cloned and sequenced, and six small interfering RNAs (siRNAs) targeting the tyrosine kinase region were developed. The most effective siRNA as well as an EGFR specific tyrosine kinase inhibitor, gefitinib, was then used on a feline SCC cell line (SCCF1), and the effect of EGFR targeting alone, or in combination with irradiation, on the cell line was determined. The majority of the biopsy samples were labelled positively for EGFR and Ki67, and high proliferation corresponded with poor prognosis. The siRNA caused reduction in EGFR mRNA by Real-Time Polymerase Chain Reaction and protein levels as assessed by western blot analysis. Reduced cell proliferation and migration were also observed by proliferation assays and scratch assays respectively. Combining EGFR knockdown with irradiation caused an additive effect on the ability of the cell line to form colonies. These results support the role of EGFR as a potential therapeutic target in FOSCCs.

Human gliomas represent the most common primary brain tumours in adults. According to World Health Organization classification, gliomas are divided into astrocytomas with four grades (I, II, III, and IV), oligodendrogliomas with two grades (II and III), and oligoastrocytomas with two grades (II and III) based on the tumor cell phenotype. Pathological classification remains controversial due to the lack of specific immunohistochemical biomarker to recognize gliomas. Also, due to their natural propriety to infiltrate the normal parenchyma and to migrate far from the first location, total surgical resection remains often impossible then adjuvant treatment is needed. The established therapies for gliomas include surgery, radiotherapy and chemotherapy. Despite this arsenal of therapies, median survival of the most malignant grade glioblastoma is approximately 15 months. This is why the attempts to better understand the molecular biology of gliomas in the aim to define new molecular targets is a holy grail. Using conventional and molecular cytogenetic approaches and molecular genetic methods, we have investigated patients bearing gliomas and followed at CHUS. Our results display that the codeletion 1p/19q (1p-/19-) is not only a prognostic and predictive biomarker of oligodendrogliomas but also a diagnostic tool of this tumor. Our study has allowed us to build a glioma-bank containing around 150 samples of patients, which we continued to populate. We have also defined the cut-off positivity of FISH on touch preparation slides, which is 20%. In addition, we have developed a new, fast and reliable method to retrieve the 1p-/19q- in all samples 24 hours after sampling. This method was transferred to the clinical lab. Furthermore, we have successfully cultured the brain tumor samples and analyzed their caryotypes. This has permitted us to discover a new alternative translocation, which is responsable of 1p deletion in one oligoastrocytoma case. Since glioblastoma is characterized by genomic instability, and telomere disruption is a main cause of genomic instability, we investigated the nuclear telomere architecture in this type of tumour. We found that nuclear telomeric architecture could be a biomarker of glioblastoma since it can subdivide glioblastoma patients in three categories with significantly different outcomes and time to progression. We used high-throughput methods (one manual and one semi-automatic) to characterize the nuclear telomeric architecture in glioblastoma. The semi-automatic method can be transfer to the clinical lab since it can deliver the results nine hours after sampling. Then, this approach is usable to monitor this tumor and to evaluate the impact of different treatment options. We need to search the putative tumor suppressor genes on chromosomes 1 and 19. Furthermore, trying to understand the molecular basis of nuclear telomeric architecture will bring up new molecular target therapies.

No description available.

La Fibrose Kystique, causée par des mutations du canal CFTR, mène à la dysfonction du transport des fluides et des ions causant la déshydratation du liquide de surface des voies aériennes et ainsi une défaillance de la clairance mucocilliaire. Ce défaut entraine l’accumulation et l’épaississement du mucus au niveau des bronches qui devient alors un environnement idéal pour le développement d’infections chroniques et d’inflammation qui sont associées à la destruction progressive de l’épithélium chez les patients Fibrose Kystique. Même si leur rôle dans les processus lésionnels est très bien connu, l’impact de médiateurs inflammatoires sur la capacité de réparation ne l’est cependant pas. L’objectif de ma maitrise était donc d’étudier la régulation des mécanismes de réparation de l’épithélium bronchique sain et Fibrose Kystique par le facteur de nécrose tumoral (TNF)-alpha, une cytokine pro-inflammatoire cruciale dans l’initiation et la propagation de la réponse inflammatoire chez les patients FK. À l’aide d’un modèle de plaies mécaniques, nous avons montré que le TNF-alpha stimule la réparation de l’épithélium bronchique sain (NuLi-1) et Fibrose Kystique (CuFi-1). De façon surprenante, l’exposition chronique au TNF-alpha augmente cette stimulation tout comme le taux de migration cellulaire pendant la réparation. Cette augmentation de réparation semble être médiée par l’activation de la métalloprotéinase MMP-9, la relâche d’EGF par les cellules épithéliales et ainsi l’activation de la voie d’EGFR. De plus, l’activation de la réparation par le TNF-alpha semble aussi impliquer l’activation des canaux K+, dont nous avons démontré le rôle important dans la réparation. Contrairement à son effet sur la migration cellulaire et sur la réparation, le TNF-alpha diminue la prolifération cellulaire. En somme, en plus de son rôle dans les processus lésionnels, le TNF-alpha semble avoir un rôle complexe dans les processus de réparation puisqu’il stimule la migration et ralentit la prolifération cellulaire. / Cystic fibrosis (CF) pathology, caused by mutations of cftr gene, leads to ion and fluid transport dysfunction that results in mucus thickening and accumulation in the airways. This mucus accumulation promotes bacterial infection and airway inflammation associated with progressive airway epithelial damage in CF patients, unfortunately leading to respiratory failure. However, the effect of inflammatory products on the repair capacity of respiratory epithelia is unclear. Thus, the objective of my project was to study the regulation of normal and CF bronchial epithelial repair mechanisms by tumor necrosis factor-alpha (TNF)-alpha, a major component of inflammation initiation and propagation in CF. With a wound healing model, we observed that TNF-alpha stimulated the non-CF (NuLi-1) and CF (CuFi-1) bronchial wound healing rate. Surprisingly, chronic exposure to TNF-alpha enhanced this stimulation as well as the migration rate during repair. This wound healing rate stimulation by TNF-alpha seems to be due to metalloproteinase MMP-9 activation, EGF shedding by epithelial cells and subsequent EGFR transactivation. Furthermore, we recently reported a crucial relationship between the EGF response and K+ channel function, both controlling bronchial repair. We now show that TNF-alpha wound healing stimulation also implicated KvLQT1 and KATP currents activation. In contrast to its effect on cell migration, TNF-alpha downregulate cell proliferation. Thus, in addition to its recognized role in the inflammatory response leading to epithelial injury, TNF-a could exert complex actions on repair mechanisms of CF airway epithelia by upregulating cell migration while downregulating proliferation.

Glioblastoma Multiforme (GBM) is an aggressive and fatal cancer of the brain. It is characterized with augmented morbidity and elusion to therapies due in part to the incessant infiltration and spread of tumor cells in normal brain. We investigated the function of Matrix metalloproteinase-1, an important enzyme noted to be responsible for invasion in other cancers, in GBM and its regulation by epidermal growth factor receptor (EGFR) signaling. Previous studies from our laboratory demonstrated elevated levels of MMP-1 in GBM. Further studies indicated the involvement of MMP-1 in GBM invasion. The GBM cell lines T98G, U251MG and U87MG were used for this study. In T98G cell lines, inhibition of MMP-1 by siRNA significantly suppressed basal in vitro invasion without impacting cell viability. The over-expression of MMP-1 was accomplished in U251MG and U87MG using the mammalian expression vector, pIRES, encoding full length MMP-1 cDNA. The MMP-1 over-expressing U251MG and U87MG cells exhibited significantly enhanced invasion in vitro with no modification in the cell proliferation rates. A majority of GBM patients present defective EGFR signaling due to over-expression, amplification or mutation in the receptor. MMP-1 is known to be up-regulated by various stimulatory agents including growth factors. We examined the regulation of MMP-1 by EGFR activation and observed the induction of MMP-1 after EGF treatment. Inhibition of the receptor by pharmaceutic inhibitor treatment and genetic approaches led to reduction in MMP-1 levels. We also observed that this regulation is primarily mediated by the downstream MAPK pathway. Inhibition of MAPK and not PI3K pathway resulted in diminished MMP-1 protein levels even in the presence of EGF. These studies demonstrate the importance of the EGFR-MAPK signaling pathway in the induction of MMP-1 in glioma cell lines. In addition, MMP-1 plays a role in glioma cell invasion in vitro. These results along with the reports of MMP-1 over-expression in GBM warrant future studies examining the function of MMP-1 in vivo.

Dendrimers are a class of polymers with a highly branched, three-dimensional architecture composed of an initiator core, several interior layers of repeating units and multiple surface groups. They have been recognized as the most versatile compositionally and structurally controlled nanoscale building blocks throughout the fields of engineering, materials science, chemistry, and biology, and they have been widely investigated for drug and gene delivery. Polyamidoamine (PAMAM) dendrimers have inherent properties for gene delivery because of their high buffering capacity, polycationic surface and numerous surface groups for biofunctionlization. This dissertation is organized into four independent sections. The first section investigates a series of polyamidoamine-polyethylene glycol-poly (D,L-lactide) (G3.0- PEG1500-PDLLA, G3.0-PEG6000-PDLLA, and G3.0-PEG12000-PDLLA) for gene delivery. Western Blot, fluorescence microscopy and flow cytometry were used as analysis methods. According to gene transfection studies, G3.0-PEG1500-PDLLA has been shown to be capable of inducing higher gene expression than the parent dendrimer compared to unmodified dendrimer, G3.0-PEG6000-PDLLA and G3.0-PEG12000- PDLLA. The second section aims to evaluate an epidermal growth factor (EGF)-containing PAMAM G4.0 dendrimer vector labeled with quantum dots for targeted imaging and nucleic acid delivery. Targeting efficiency, cell viability, proliferation, and intracellular signal transduction were evaluated. We found that EGF-conjugated dendrimers did not stimulate growth of epidermal growth factor receptor (EGFR)-expressing cells at the selected concentration. Consistent with this, minimal stimulation of post-receptor signaling pathways was observed. These nanoparticles can localize within cells that express the EGFR in a receptor-dependent manner, whereas uptake into cells lacking the receptor was low. Vimentin short hairpin RNA (shVIM) and yellow fluorescent protein (YFP) small interfering RNA (siRNA) were used to test the delivery and transfection efficiency of the constructed targeted vector. Significant knockdown of expression was observed, indicating that this vector is useful for introduction of nucleic acids or drugs into cells by a receptor-targeted mechanism. The third section introduces PEGylated polyamidoamine (PAMAM) dendrimer G4.0 conjugates with a novel bis-aryl hydrazone (BAH) linkage for gene delivery. It was found that the incorporation of BAH linkages into the vector significantly enhanced the buffering capacity of the vector with a high degree of PEGylation. According to gene transfection studies, this new vector has been shown to be capable of both transfecting more cells and inducing higher gene expression than the parent dendrimer. This work demonstrates that the use of the BAH linkage in coupling of PEG to the dendrimer helps maintain or increase the buffering capacity of the functionalized dendrimer and results in enhanced transfection. In the fourth section, we explored PAMAM dendrimer G4.5 as the underlying carrier to construct central nervous system (CNS) therapeutic nanoparticles and tested the buccal mucosa as an alternative absorption site for administration of the dendritic nanoparticles. Opioid peptide DPDPE was chosen as a model CNS drug. It was coupled to PAMAM dendrimer G4.5 with PEG or with PEG and transferrin receptor monoclonal antibody OX26. The therapeutic dendritic nanoparticles labeled with 5-(aminoacetamido) fluorescein (AAF) or fluorescein isothiocyanate (FITC) were studied for transbuccal transport using a vertical Franz diffusion cell system mounted with porcine buccal mucosa. Coadministration of bile salt sodium glycodeoxycholate (NaGDC) or application of mucoadhesive gelatin/PEG semi-interpenetrating network (sIPN) enhanced the permeability of dendritic nanoparticles by multiple folds. These results indicate that transbuccal delivery is a possible route for administration of CNS therapeutic nanoparticles. In summary, enhanced nucleic acids delivery by biofunctionalized PAMAM dendrimers was demonstrated. Transbuccal delivery of CNS therapeutic dendritic nanoparticles was demonstrated. These vectors will be useful in gene and drug delivery and could be extended to covalently conjugate other functional moieties for gene and drug delivery.