Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The development of targeted therapy has contributed tremendously to the treatment of patients with cancer. The use of highly specific affinity proteins to target cancer cells has become a standard in treatment strategies for several different cancers. In light of this, many cancer cell markers are investigated for their potential use in diagnostics and therapy. One such marker is the human epidermal growth factor receptor 3, HER3. It has been established as an important contributor to many cancer types. The function of HER3 is to relay cell growth signals from outside of the cell to the inside. Interfering with- and inhibit- ing the function of HER3 has emerged as an interesting strategy for cancer therapeutics. The studies presented in this thesis aim to target HER3 with small, engineered affinity domain proteins for therapeutic purposes. Monomeric affibody molecules have previously been engineered to bind and inhibit HER3 in vitro. Due to the relatively low expression of HER3, an increase in valency appears promising to strengthen the therapeutic potential. Affibody molecules targeting the receptor were thus linked to form bivalent and bispecific constructs and evaluated both in vitro and in vivo. In the first study of this thesis affibody molecules specific for HER3 and HER2 were fused to an albumin binding domain to form bivalent and bispecific construct. The constructs inhibited ligand-induced receptor phos- phorylation of both HER2 and HER3 more efficiently than monomeric affibody molecules. A second approach to enhance the potential of affibody molecules in tumor targeting is described in the second study, where monomeric HER3-binding affibody molecules were engineered to increase their affinity for HER3. The resulting variants showed a 20-fold in- creased affinity and higher capacity to inhibit cancer cell growth. Combining the findings of the first two studies, the third study describes the evaluation of a HER3-targeting bivalent affibody construct for potential application as a therapeutic. Here, the bivalent construct inhibited cancer cell growth in vitro and was found to slow down tumor growth in mice, while being well tolerated and showing no visible toxicity. The fourth study built upon these findings and compares a very similar bivalent construct to the clinically-investigated HER3-specific monoclonal antibody seribantumab. The affibody construct showed very comparable efficacy with the antibody in terms of decreasing tumor growth rate and ex- tending mouse survival. Collectively, these works describe for the first time the use of alternative affinity protein constructs with therapeutic potential targeting HER3. / <p>QC 20170330</p>

INTRODUÇÃO: O câncer do pâncreas apresenta taxas anuais de mortalidade e incidência muito semelhantes, sendo uma das principais causas de morte por câncer no mundo. A agressividade do tumor e o retardo no seu diagnóstico são considerados responsáveis pela sua alta letalidade. O tratamento adjuvante convencional aumenta pouco a sobrevida a longo prazo e a terapia-alvo pode ser uma alternativa ao tratamento deste tipo de tumor. OBJETIVO: O objetivo do presente estudo é avaliar a expressão do receptor do fator de crescimento epitelial e seu eventual valor prognóstico em pacientes portadores de adenocarcinoma pancreático submetidos à ressecção cirúrgica. MÉTODO: Foram estudados retrospectivamente 88 pacientes portadores de adenocarcinoma pancreático operados no Serviço de Cirurgia de Vias Biliares e Pâncreas do HC-FMUSP e no Departamento de Cirurgia Abdominal Hospital A.C. Camargo no período de 1990 a 2006. RESULTADOS: Quarenta e sete (53,4%) pacientes do sexo feminino e 41 (46,6%) do masculino com idade mediana de 60 anos. As cirurgias realizadas foram duodenopancreatectomia com preservação do piloro (55,1%), gastroduodenopancreatectomia (34,8%), pancreatectomia corpo-caudal (6,8%) e gastroduodenopancreatectomia total (2,3%). A ressecção venosa portal foi realizada em 12 pacientes (13,5%). O tamanho tumoral médio foi de 3,75cm. Invasão vascular esteve presente em 31% dos casos e neural em 88,5%. A margem de ressecção estava comprometida em 33 pacientes (37,5%). Cinco (5,7%) pacientes eram do estádio IA, 15(17%) do IB, 19(21,6%) do IIA, 47(53,4) do IIB e dois (2,3%) do III.Observou-se diferença na expressão de EGFR na membrana celular entre o tecido tumoral e o tecido não tumoral (p=0,004); entre o tecido metastático linfonodal e o tecido não tumoral (p=0,02) mas não houve diferença quanto à sua expressão quando comparamos o tecido tumoral e o tecido metastático linfonodal (p=0,28). Dentre as variáveis clínicas e patológicas estudadas, observou-se diferença de expressão do EGFR entre os gêneros feminino e masculino (p=0,03), não havendo diferenças entre as outras variáveis. A sobrevida mediana global foi de 22,9 meses. A sobrevida cumulativa global em 1 ano, 3 anos e 5 anos foi de 48%, 20% e 18%, respectivamente. As sobrevidas cumulativas em 1 ano, 3 anos e 5 anos foram 77%, 30% e 8% no grupo sem expressão do EGFR na membrana tumoral versus 46%, 8% e 0% respectivamente no grupo com expressão do EGFR na membrana celular do tumor. Na análise univariada, as seguintes variáveis estiveram associadas a menor sobrevida: sexo masculino, ressecção venosa portal, invasão peri-neural, e vascular, invasão do tecido peri-pancreático, acometimento da margem de ressecção pancreática e expressão positiva de EGFR no tecido tumoral. Na análise multivariada, os fatores associados à sobrevida menor foram: gênero masculino, ressecção venosa portal, invasão vascular e invasão peri-neural. CONCLUSÃO: A expressão do EGFR na membrana celular é significativamente maior no tecido tumoral que no tecido pancreático não tumoral. A expressão do EGFR na membrana celular do tecido tumoral está associada a pior prognóstico (menor sobrevida). / INTRODUCTION: Pancreatic cancer is one of the main cancer related deaths in the world and its incidence is similar to its mortality. Biological aggressiveness and delayed diagnosis are a major concern. Adjuvant treatment has little impact on survival and the expression of potential target molecules has been undertaken in order to increase survival. OBJECTIVE: The aim of the present study is to study the expression of EGFR and its potential prognostic role in tumor, non-tumor and metastatic lymph node tissues of patient with pancreatic adenocarcinoma treated with surgical resection. MATHERIAL AND METHODS: Eighty eight patients with pancreatic adenocarcinoma operated at Serviço de Cirurgia de Vias Biliares e Pâncreas do HC-FMUSP and Departamento de Cirurgia Abdominal do Hospital A.C.Camargo were retrospectively studied between 1990 and 2003. RESULTS: Forty seven females (53,4%) and 41 males (46,6%) with median age of 60 years were studied. Pylorus preserving duodenopancreatectomy was performed in 55%, classical duodenopancreatectomy in 34,8%, distal pancreatectomy in 6,8% and total pancreatectomy in 2,3%. Portal vein resection was performed in 12 patients (13,5%). Mean tumor size was 3,75cm. Vascular and neural invasion were present in 31% and 88,5%, respectively. Positive surgical margin was present in 33 (37,5%) patients. Five (5,7%) patients were stage IA, 15(17%) stages IB, 19(21,6%) stages IIA, 47(53,4%) stages IIB and two (2,3%) stages III. There were difference in the membrane expression of EGFR between tumor and non tumor pancreatic tissue (p=0,004); between metastatic lymph node and non tumor pancreatic tissue (p=0,02); but there were no difference between tumor and metastatic lymph node tissue (p=0,28). Median survival time was 22,9 months. Cumulative one, three and five years survival were 48%, 20% and 18%. Cumulative survival at 1, 3 and 5 years were 77%, 30% and 8% in patients with negative expression of EGFR in tumor membrane and 46%, 8% and 0%, respectively in patients with positive EGFR expression in tumoral membrane. Univariate analysis showed that male gender, portal vein resection, neural, vascular and peri-pancreatic invasion invasion, positive surgical margin and positive membrane EGFR expression in tumoral tissue were correlated with poor survival. Multivariate analysis showed that male gender, portal vein resection, vascular invasion and peri-neural invasion are associated with lower survival after resection. CONCLUSION: EGFR membrane expression is different between tumor tissue and non tumor pancreatic tissue. EGFR membrane expression in tumoral tissue was associated with worst survival.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 154 pages. Includes vita. Includes bibliographical references.

A novel calmodulin (CaM)-binding cysteine-rich protein from Dictyostelium, cyrA, with epidermal growth factor-like (EGFL) repeats was discovered and characterized. Calcium-dependent and –independent CaM-binding was verified. Western blots show that full length cyrA is detected constitutively throughout development. Analyses of the extracellular medium reveal that cyrA is cleaved and that the fragments containing the N-terminus are secreted early in development, while those containing the C-terminus are secreted later. In support of this, GFP and immunohistochemistry studies reveal that cyrA localizes to the endoplasmic reticulum and secretory vesicles of vegetative cells, and to the extracellular matrix (slime sheath) of migrating slugs. The addition of EGFL1 peptides enhanced cell motility and cAMP-mediated chemotaxis. Finally, cyrA cleavage is regulated by extracellular Dictyostelium CaM and by the extracellular EGFL repeats. In total the data suggest that cyrA is a true matricellular protein that mediates cell motility during multicellular development.

A novel calmodulin (CaM)-binding cysteine-rich protein from Dictyostelium, cyrA, with epidermal growth factor-like (EGFL) repeats was discovered and characterized. Calcium-dependent and –independent CaM-binding was verified. Western blots show that full length cyrA is detected constitutively throughout development. Analyses of the extracellular medium reveal that cyrA is cleaved and that the fragments containing the N-terminus are secreted early in development, while those containing the C-terminus are secreted later. In support of this, GFP and immunohistochemistry studies reveal that cyrA localizes to the endoplasmic reticulum and secretory vesicles of vegetative cells, and to the extracellular matrix (slime sheath) of migrating slugs. The addition of EGFL1 peptides enhanced cell motility and cAMP-mediated chemotaxis. Finally, cyrA cleavage is regulated by extracellular Dictyostelium CaM and by the extracellular EGFL repeats. In total the data suggest that cyrA is a true matricellular protein that mediates cell motility during multicellular development.

The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase integral in regulating cell growth, survival, and migration. EGFR signaling, which is dependent on localization of the receptor within lipid rafts, is often hijacked during colon tumorigenesis. Previous work has found that docosahexaenoic acid (DHA) is protective against colon cancer. This fatty acid is proposed to function in part by perturbing lipid rafts and thereby altering cell signaling. The overall objective of this work was to determine whether DHA alters EGFR function and signaling. We assessed EGFR localization and ligand-induced phosphorylation in YAMC cells treated with fatty acids. We found that DHA reduced the localization of EGFR to lipid rafts. Concomitant with altering receptor localization, DHA was found to increase EGFR phosphorylation. However, DHA paradoxically suppressed EGFR signal transduction. We found that DHA uniquely altered EGFR activity, and other long chain polyunsaturated fatty acid did not exert the same effect. We additionally observed similar effects on EGFR activation and signaling by feeding mice a diet enriched in fish oil (high in DHA), and this was attendant with reduced colon tumorigenesis. We next probed the mechanism by which DHA enhances EGFR phosphorylation. We found that DHA facilitates receptor dimerization to increase phosphorylation. We additionally identified Ras activation as the site of perturbation of signal transduction. DHA suppressed signal transduction by both changing the localization of EGFR within the plasma membrane and increasing receptor endocytosis and degradation. Lastly, we extended our observations into a wounding model. Although DHA uniquely altered ligand-stimulated EGFR activity, both DHA and EPA altered EGFR transactivation and signaling upon injury. This culminated in reduced wound healing in DHA and EPA treated cells. In an animal model, we found that diets enriched in either DHA or EPA altered EGFR signaling in the colonocytes of wounded animals. Overall, we found that DHA modifies EGFR signaling, which can be beneficial or detrimental for health depending on the disease state of an individual. These data help elucidate a mechanism by which DHA protects against colon cancer, as well as indicating a potential downside of n-3 PUFA therapy.

In this study, we hypothesize that human acidic mammalian chitinase (AMCase) binds and is regulated by the epidermal growth factor receptor (EGFR), and that AMCase interacts with Galectin-3 (Gal-3) to mediate anti-apoptotic functions. We further hypothesize that asthma-associated polymorphisms of AMCase alter chitinase activity and modulate anti-apoptotic effects. We investigated the interactions between AMCase, Gal-3 and EGFR by establishing binding and co-expression in vitro; apoptotic effects were evaluated via Annexin V/Propidium Iodide staining. Molecular cloning was performed to generate single nucleotide polymorphisms (SNPs) of AMCase associated with asthma. Our data showed that co-expression of AMCase and EGFR induces chitinase activity; we found that AMCase and Gal-3 bind each other in vitro, and that they co-localize in the cytoplasm of cells. Co-transfection of AMCase and Gal-3 demonstrates greater anti-apoptotic effect than Gal-3 alone, while recombinant Gal-3 induces apoptosis, which is not blocked by incubation with recombinant AMCase. From these data, we conclude that AMCase is regulated by EGFR, and that AMCase and Gal-3 physically interact, however contrary to our hypothesis, the anti-apoptotic effects of AMCase are unlikely to be mediated by Gal-3. Further exploration of this pathway using SNP constructs generated in this study will shed light on the mechanism of AMCase in asthma.

L'hypervascularisation artérielle bronchique des dilatations des bronches contribue à l'afflux local de cellules inflammatoires et de protéines plasmatiques et favorise la survenue de saignements bronchiques. Les mécanismes de l'angiogénèse des vaisseaux bronchiques dans les dilatations des bronches sont peu connus, mais pourraient impliquer la voie du facteur de croissance endothélial (VEGF)-A.Nous avons émis l'hypothèse que l'infection bronchique dans les dilatations des bronches contribue à l'angiogénèse des vaisseaux bronchiques. Nous avons développé chez la souris un modèle d'infection bronchique persistante par l'instillation intratrachéale de billes d'agarose contenant du Pseudomonas aeruginosa. Nos résultats indiquent que l'infection bactérienne provoque l'angiogénèse des vaisseaux péribronchiques en 7 jours. P. aeruginosa induit la synthèse de VEGF-A par l'épithélium respiratoire in vitro et chez la souris par l'activation du récepteur de l'epidermal growth factor (EGF).Nous avons ensuite évalué l'effet de la perte de fonction de CFTR, l'anomalie caractéristique de la mucoviscidose (une cause génétique de dilatation des bronches), sur l'angiogénèse bronchique et l'expression de facteurs pro-angiogéniques. Ces études ont été menées à partir de poumons de patients mucoviscidosiques, chez des souris mutées pour le gène cftr et par inhibition de CFTR sur des cultures de cellules épithéliales.Nos données indiquent que l'infection bronchique contribue à l'angiogénèse péribronchique, qui nécessiterait une communication épithélium/endothélium. L'épithélium bronchique de la mucoviscidose est dans un état pro-angiogénique en l'absence d'infection.

国立情報学研究所で電子化したコンテンツを使用している。