Thioredoxin-1 (Trx-1) is a redox protein that is overexpressed in many tumors where it is associated with tumor growth, inhibited apoptosis and decreased patient survival. Through redox reactions, Trx-1 is able to reduce a number of proteins including transcription factors. Sp1 activation has been implicated in the regulation of many genes involved in cellular growth and survival and its overexpression in certain cancer correlates with decreased patient survival. We demonstrate that Trx-1 is able to activate Sp1 in a redox dependent manner. Trx-1 overexpression increases Sp1 transactivation and DNA binding whereas a redox inactive Trx-1 has no effect on Sp1 DNA binding.Sp1 has been implicated in vascular endothelial growth factor regulation and we have shown that Trx-1 expression results in increased hypoxic VEGF expression and increased tumor permeability in vivo. Trx-1 overexpression results in an increase in VEGF expression that is dependent upon Sp1, as inhibition of Sp1 expression with siRNA prevented the induction of VEGF expression by Trx-1. These results suggest that Trx-1 increases VEGF expression under normoxic conditions through a redox dependent increase in the DNA binding of the Sp1 transcription factor. VEGF regulation by Sp1 could increase angiogenesis in relatively perfused areas contributing to the stimulation of tumor growth by Trx-1.We hypothesized that Trx-1 regulation of Sp1 may be part of the mechanism of Trx-1 induction of cellular growth. Sp1 regulates many genes involved in cellular growth including epidermal growth factor receptor (EGFR) and insulin-like growth factor I receptor (IGF-IR). These two growth factor receptors are important for cellular growth and have been shown to be important therapeutic targets for cancer treatment. We report that treatment with the Trx-1 inhibitor PX-12 results in decreased Sp1 DNA binding as well as decreased Sp1 activation and transactivation of VEGF, EGFR, and IGF-IR. These results indicate that Trx-1 promotes cellular growth and survival, in part, through the redox regulation of Sp1 responsive growth genes EGFR and IGF-IR. Inhibition of Trx-1, via PX-12, results in a decrease in EGFR and IGF-IR expression and suggests a new mechanism by which Trx-1 inhibition is clinically effective for treating cancer.

PHIP1 is a novel downstream transcriptional co-regulator of insulin-like growth factor-I receptor (IGF-IR), a tyrosine kinase receptor that is often elevated and autophosphorylated in breast cancer. In this study, I show that PHIP1 is upregulated in MCF10A cells stably overexpressing IGF-IR signaling components and that knock-down of PHIP1 significantly inhibits breast cancer cell proliferation by inducing transcriptional upregulation of p21 and downregulation of cyclin D2. I also show that stable overexpression of PHIP1 in MCF10A cells can lead to its proteasomal degradation. Together, our data indicate that PHIP1 is implicated in breast cancer cell growth and suggest a number of avenues that await exciting discovery.

Tumorerkrankungen stellen in der westlichen Welt eines der wichtigsten Gesundheitsprobleme dar. Das kolorektale Karzinom ist dabei die dritthäufigste Tumorneuerkrankung. Bei fortgeschrittenem Krankheitsverlauf wird zumeist eine kombinierte Radiochemotherapie durchgeführt, bei der zusätzlich zur Bestrahlung Zytostatika wie 5-Fluoruracil oder Oxaliplatin verabreicht werden. Da die eingesetzten Zytostatika nicht ausschließlich gegen Tumorzellen wirken, führt der Einsatz dieser oft zu massiven Nebenwirkungen wie Magen- und Darmproblemen, Myelosuppression und Haarausfall. Neue Therapieansätze versuchen daher Ziele in die Behandlung mit aufzunehmen die stärker karzinomspezifisch sind, wie z.B. verschiedenen Rezeptortyrosinkinasen. Viele Rezeptortyrosinkinasen und deren Liganden liegen im Tumor und umliegenden Gewebe oft dereguliert vor und spielen eine wichtige Rolle bei der Regulierung des Tumorwachstums, der Tumorangiogenese und der Metastasenbildung. In dieser Arbeit konnte für die drei kolorektalen Karzinomzelllinien DLD-1, SW837 und Caco 2 gezeigt werden, dass die gleichzeitige Inhibition des Insulin-like Growth Factor-I Receptor (IGF-IR) und des Epidermal Growth Factor Receptor (EGFR) mit den Tyrosinkinaseinhibitoren AEW-541 (IGF-IR-Inhibitor) und Erlotinib (EGFR-Inhibitor) in vitro zu einem deutlich verstärkten Therapieeffekt der 5-Fluoruracil-basierten Radiochemotherapie führt. Für Xenografttumore der Zelllinie SW837 konnte dieser Effekt auch in vivo bestätigt werden. Mit Hilfe der Co Immunpräzipitation und eines Proximity Ligation Assays konnten in den Kolonkarzinomzelllinien SW480 und DLD-1 Hybridrezeptoren zwischen dem EGFR und dem IGF-IR nachgewiesen werden. Zusätzlich konnte gezeigt werden, dass eine Ligandenstimulation der Rezeptoren zu einer vermehrten EGFR/IGF-IR-Hybridrezeptorbildung führt. Weitere Analysen zeigten, dass für die induzierte Heterodimerisierung beide Liganden notwendig sind und beide Rezeptoren funktionsfähig sein müssen. Mit Hilfe des Proximity Ligation Assays konnten IGF-IR/EGFR-Hybridrezeptoren auch in humanen Rektumtumoren nachgewiesen werden. Im letzten Teil der Arbeit wurde die Bedeutung des Platelet-derived Growth Factor Receptor β (PDGFR-β) in kolorektalen Karzinomzellen untersucht. In SW480- und DLD-1-Zellen führte die Inhibition des PDGFR-β mit Hilfe von spezifischer siRNA zu einer, über den PI3K-Signalweg vermittelten, moderat verminderten Proliferationsrate. Die Verwendung des PDGFR-β-Inhibitors Ki11502 führte in den Zelllinien zu einem starken Rückgang in der Proliferationsrate und zu Veränderungen im Zellzyklus der Zellen. Diese wurden durch eine verminderte Cyclin-B1-Expression hervorgerufen. Weitere Analysen zeigten, dass der Inhibitor Ki11502 neben dem PDGFR-β auch den Rezeptor cKIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) und die Zellmembran-assoziierte zytoplasmatische Tyrosinkinase SRC (v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog) inhibiert.

A distrofia muscular de Duchenne (DMD) é uma doença de caráter hereditário recessivo ligado ao cromossomo X, que determina a ausência da distrofia, a responsável pela progressiva degenaração muscular observada no DMD. Embora não expresse o fenótipo, a camundongo mdx apresenta a ausência da distrofia e o mais comum modelo animal experimental para estudar as repercursões da DMD em muitos orgãos. Este estudo foi realizado na cartilagem do processo condilar da mandíbula de mdx, um importante sítio de crescimento craniofacial. Assim, o PC dos mdx de 4 (G1) e 10 (G2) semanas e dos respectivos controles (camundongos c57BL/10 mice) foram avaliados usando as técnicas de microscopia de luz (H.E, Picrosirius e Safranina-O) e de imunohistoquímica (IGF e IGF-IR). Em ambos os grupos, não houve diferença estatísticamente significante na área do PC e comparação aos controles. O número de e a área dos condrócitos, bem como, a quantidade de matriz extracelular (MEC) forma menores nos grupos mdx. A imunorreatividade para ambos, IGF-I e IGF-IR, proporcionalmente maiores nos grupos mdx. Os dados quantitativos e predominância do colágeno tipo I nos grupos mdx, sugere um processo precoce de envelhecimento na cartilagem do PC desses animais. / The duchenne muscular dystrophy (DMD) is a recessive hereditary disease linked to X-chromossome that determines teh dystrophin abstence, the responsible for progressive muscle degeneration observed in DMD. Although not exhibit the phenotype, the MDX mouse reveal abstence of dystrophin and is the most common experimental animal model for DMD studies in many organs. This study was performed in the articular cartilage of the mandibular condylar process (PC) of MDX, an important site of craniofacial growth. Thus the PC of MDX and respective controls (C57BL/10 mice) were evaluated at the ages of 4 (G1) and 10 (G2) weeks using ligth microscopy (H.E, Picrosirius e Safranin-O) and immunohistochemical (IGF-I e IGF-IR) tecniques. In both groups, there was no statistical significant difference in PC area of the mdx and the respective controls. The number and area of the chondrocytes, as well as the amout of extracellular matrix (MEC) were lower in MDX groups. The immunoreactivity for both, IGF-I and IGF-IR, were propostionally higher im MDX groups. The quantitative data and the predominance of collagen type i fibers in the MDX groups suggest a premature aging process of the PC in these animals.

A distrofia muscular de Duchenne (DMD) é uma doença de caráter hereditário recessivo ligado ao cromossomo X, que determina a ausência da distrofia, a responsável pela progressiva degenaração muscular observada no DMD. Embora não expresse o fenótipo, a camundongo mdx apresenta a ausência da distrofia e o mais comum modelo animal experimental para estudar as repercursões da DMD em muitos orgãos. Este estudo foi realizado na cartilagem do processo condilar da mandíbula de mdx, um importante sítio de crescimento craniofacial. Assim, o PC dos mdx de 4 (G1) e 10 (G2) semanas e dos respectivos controles (camundongos c57BL/10 mice) foram avaliados usando as técnicas de microscopia de luz (H.E, Picrosirius e Safranina-O) e de imunohistoquímica (IGF e IGF-IR). Em ambos os grupos, não houve diferença estatísticamente significante na área do PC e comparação aos controles. O número de e a área dos condrócitos, bem como, a quantidade de matriz extracelular (MEC) forma menores nos grupos mdx. A imunorreatividade para ambos, IGF-I e IGF-IR, proporcionalmente maiores nos grupos mdx. Os dados quantitativos e predominância do colágeno tipo I nos grupos mdx, sugere um processo precoce de envelhecimento na cartilagem do PC desses animais. / The duchenne muscular dystrophy (DMD) is a recessive hereditary disease linked to X-chromossome that determines teh dystrophin abstence, the responsible for progressive muscle degeneration observed in DMD. Although not exhibit the phenotype, the MDX mouse reveal abstence of dystrophin and is the most common experimental animal model for DMD studies in many organs. This study was performed in the articular cartilage of the mandibular condylar process (PC) of MDX, an important site of craniofacial growth. Thus the PC of MDX and respective controls (C57BL/10 mice) were evaluated at the ages of 4 (G1) and 10 (G2) weeks using ligth microscopy (H.E, Picrosirius e Safranin-O) and immunohistochemical (IGF-I e IGF-IR) tecniques. In both groups, there was no statistical significant difference in PC area of the mdx and the respective controls. The number and area of the chondrocytes, as well as the amout of extracellular matrix (MEC) were lower in MDX groups. The immunoreactivity for both, IGF-I and IGF-IR, were propostionally higher im MDX groups. The quantitative data and the predominance of collagen type i fibers in the MDX groups suggest a premature aging process of the PC in these animals.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Members of the insulin-like growth factor (IGF) family are mitogenic growth factors which have been shown to play critical roles in both normal growth and development, and tumour biology. The IGF system is complex and the biological effects of the IGFs are determined by diverse interactions between many molecules, including interactions with the extracellular matrix (ECM). Recent observations have demonstrated that IGFs can associate with the ECM protein vitronectin (VN) and this interaction can modulate IGF-stimulated biological functions. It has been demonstrated previously that IGF-II can bind directly to VN, while IGF-I associates with VN indirectly via the involvement of IGF-binding proteins (IGFBPs) -2, -3, -4 and -5. As the IGF system plays important roles in both normal breast development and in the transformation and progression of breast cancer, this study aimed to describe the effects of substrate-bound IGF-I:IGFBP:VN complexes on breast cell functions and to dissect the mechanisms underlying these responses. The studies reported in this thesis demonstrate that substrate-bound IGF-I:IGFBP:VN complexes, containing IGFBP-3 and IGFBP-5, are potent stimulators of proliferation and migration in the "normal", non-tumourigenic MCF-10A breast epithelial and MCF-7 breast carcinoma cell lines. Interestingly, substrate-bound IGF-I:IGFBP:VN complexes were less effective in increasing the migration of the metastatic MDA-MB-231 breast cancer cell line. This, however, is due to these cells expressing the αvβ3 integrin which can support a highly migratory phenotype independent of IGF-I-stimulation. Taken together this suggests a particularly important role for these complexes in stimulating a highly migratory phenotype in pre-invasive or poorly metastatic breast cells. Studies using IGF-I analogues were also undertaken to establish if there was a requirement for ternary complex formation and the type-1-IGF receptor (IGF-1R) in the enhanced migration responses observed. These studies determined IGF-I:IGFBP:VN-stimulated migration to be dependent upon both heterotrimeric IGF-I:IGFBP:VN complex formation and activation of the IGF-1R. Furthermore, the enhanced cellular migration was abolished upon incubation of MCF-7 and MCF-10A cells with function blocking antibodies directed at VN-binding integrins and the IGF-IR. In addition, analysis of the signal transduction pathways underlying the enhanced cell migration revealed that the complexes stimulate a transient activation of the ERK/MAPK signaling pathway, while simultaneously producing a sustained activation of the PI3-K/AKT pathway. Optimal intracellular signaling required activation of both the IGF-1R and VN-binding integrins, as antibody mediated inhibition of either receptor led to substantial decreases in both ERK/MAPK and PI3-K/AKT pathway activation. Furthermore, experiments using pharmacological inhibitors of these pathways determined a pivotal role for PI3-K/AKT activation in substrate-bound IGF-I:IGFBP:VN-stimulated cell migration. In order to confirm an important role for the PI3-K/AKT pathway in these responses, wild-type and activated-AKT was transiently overexpressed in MCF-10A cells. Overexpression of both wild-type and activated-AKT further enhanced cellular migration in response to substrate-bound IGF-I:IGFBP:VN complexes. However, these responses still required co-activation of the IGF-1R and VN-binding integrins. In an attempt to obtain a global view of the possible molecular mechanisms underpinning IGF-I:IGFBP:VN-stimulated cell migration, oligonucleotide microarrays were used to screen for candidate genes important for the observed migratory responses. The microarray studies identified 165 genes which were differentially expressed in cells migrating in response to substrate-bound IGF-I:IGFBP:VN complexes. Gene ontology and functional analysis revealed many of these genes to be significantly associated with biological functions relevant to cancer transformation and progression, including cell growth and proliferation, cell death and cellular movement. In regard to cell migration, a number of the genes identified have previously reported roles in cellular movement, migration and metastasis, which may provide future targets to augment IGF-I:IGFBP:VN-stimulated cell migration. Taken together, the studies reported throughout this thesis have provided the first mechanistic insights into the action of IGF-I:IGFBP:VN complexes and add further evidence to support the involvement of VN-binding integrins and their co-operativity with the IGF-IR in the promotion of tumour cell migration. Importantly, identifying the molecular mechanisms by which IGF:VN complexes enhance breast cell function will lead to not only a better understanding of this critical interaction, but also aid in developing diagnostic tests and therapeutics directed at treating breast cancer.

<p>Multiple myeloma (MM) is an incurable B-cell malignancy mainly localized to the bone marrow. Our aim was to examine the growth- and survival-promoting role of the IGF-IR and its downstream signaling components in MM cells to identify potential targets for therapy. </p><p>Octreotide, a somatostatin analog that has been demonstrated to interfere with the actions of IGF-I, induced growth inhibition in both IL-6-dependent and IL-6-independent MM cell lines expressing the somatostatin receptors sst2, sst3 and sst5. Additionally, a slight pro-apoptotic effect could be observed in a few cell lines. In primary MM cells octreotide induced apoptosis, an effect that was abrogated by exogenously added IGF-I, but not by IL-6.</p><p>Inhibition of IGF-I signaling in Karpas 707 cells, using either the anti-IGF-IR antibody αIR3 or the PI 3-K inhibitors LY294002 and wortmannin, increased sensitivity to apoptosis induced by dexamethasone. Exogenously added IGF-I prevented dexamethasone-induced apoptosis, an effect that could partly be mimicked by the pharmacological GSK-3β inhibitors LiCl and SB415286. Thus, we suggest the GSK-3β as an important mediator of the anti-apoptotic effects of IGF-IR signaling in MM.</p><p>Using rapamycin we selectively inhibited mTOR, a phosphoprotein downstream of the IGF-IR. In MM cell lines rapamycin induced G0/G1-arrest, an effect being associated with an increase of the cyclin-dependent kinase inhibitor p27 and a decrease of the cyclins D2, D3 and E. Interestingly, in primary MM cells rapamycin induced apoptosis. Moreover, rapamycin potentiated dexamethasone-induced apoptosis, an effect that was associated with a downregulation of the anti-apoptotic protein survivin. Strikingly, the combinatorial treatment with rapamycin and dexamethasone suppressed the anti-apoptotic effects of exogenously added IGF-I and IL-6, thus suggesting this drug-combination to be active also in vivo. </p><p>Two newly developed, selective IGF-I RTK inhibitors proved to be very effective in MM cell lines and in primary MM cells providing 50-90% growth inhibition within 48 h of incubation. The inhibitors induced massive apoptosis together with a prominent cell cycle arrest in the G2/M-phase. Importantly, the IGF-I RTK inhibitors downregulated the tyrosine phosphorylation of the IGF-IR β-chain but not of the insulin receptor β-chain. </p><p>In conclusion, the IGF-IR potently promotes growth and survival of MM cells. Therefore, interfering with the IGF-IR signaling pathway might be a suitable strategy to improve MM treatment.</p>

Multiple myeloma (MM) is an incurable B-cell malignancy mainly localized to the bone marrow. Our aim was to examine the growth- and survival-promoting role of the IGF-IR and its downstream signaling components in MM cells to identify potential targets for therapy. Octreotide, a somatostatin analog that has been demonstrated to interfere with the actions of IGF-I, induced growth inhibition in both IL-6-dependent and IL-6-independent MM cell lines expressing the somatostatin receptors sst2, sst3 and sst5. Additionally, a slight pro-apoptotic effect could be observed in a few cell lines. In primary MM cells octreotide induced apoptosis, an effect that was abrogated by exogenously added IGF-I, but not by IL-6. Inhibition of IGF-I signaling in Karpas 707 cells, using either the anti-IGF-IR antibody αIR3 or the PI 3-K inhibitors LY294002 and wortmannin, increased sensitivity to apoptosis induced by dexamethasone. Exogenously added IGF-I prevented dexamethasone-induced apoptosis, an effect that could partly be mimicked by the pharmacological GSK-3β inhibitors LiCl and SB415286. Thus, we suggest the GSK-3β as an important mediator of the anti-apoptotic effects of IGF-IR signaling in MM. Using rapamycin we selectively inhibited mTOR, a phosphoprotein downstream of the IGF-IR. In MM cell lines rapamycin induced G0/G1-arrest, an effect being associated with an increase of the cyclin-dependent kinase inhibitor p27 and a decrease of the cyclins D2, D3 and E. Interestingly, in primary MM cells rapamycin induced apoptosis. Moreover, rapamycin potentiated dexamethasone-induced apoptosis, an effect that was associated with a downregulation of the anti-apoptotic protein survivin. Strikingly, the combinatorial treatment with rapamycin and dexamethasone suppressed the anti-apoptotic effects of exogenously added IGF-I and IL-6, thus suggesting this drug-combination to be active also in vivo. Two newly developed, selective IGF-I RTK inhibitors proved to be very effective in MM cell lines and in primary MM cells providing 50-90% growth inhibition within 48 h of incubation. The inhibitors induced massive apoptosis together with a prominent cell cycle arrest in the G2/M-phase. Importantly, the IGF-I RTK inhibitors downregulated the tyrosine phosphorylation of the IGF-IR β-chain but not of the insulin receptor β-chain. In conclusion, the IGF-IR potently promotes growth and survival of MM cells. Therefore, interfering with the IGF-IR signaling pathway might be a suitable strategy to improve MM treatment.