Obesity is a multi-factorial metabolic disease, resulting in increased adipose tissue acquisition by the host. This disease increases the risk for developing co-morbidities, including Metabolic Syndrome and other disorders such as breast cancer. Obesity, and particularly abdominal obesity, is characterised by metabolic changes, including chronically elevated insulin concentrations and aberrant secretion of cytokines released from fat tissue, called adipokines. Epidemiologically, the risk of developing postmenopausal breast cancer is increased in obese individuals. The molecular link between obesity and breast cancer however is not well understood. The study presented here aimed at identifying the molecular mechanisms involved in this link, by testing the hypothesis that high insulin concentration and certain adipokines may promote breast cancer progression and/or breast cancer aetiology. A cell culture system of breast cancer cells and breast epithelial cells was employed to investigate changes in cell proliferation, activation of cell signalling pathways, cell cycle progression and apoptosis after treatment with insulin, leptin, TNF-α, adiponectin and IL-6. In MDA-MB-231 breast cancer cells, insulin treatment did not affect cell proliferation, cell cycle or apoptosis. Conversely, IR-phosphorylation, AKT-phosphorylation and ERK1/2-phosphorylation were all significantly increased. Microarray analysis indicated several important changes in gene expression with insulin treatment. Leptin treatment increased proliferation by 21%. Additional analyses of the effect of leptin indicated that neither the PI3-kinase pathway nor the MAP-kinase pathway was involved in mediating this effect. Treatment with TNF-α increased apoptosis, but did not affect cell proliferation or activation of cell signalling pathways. In MCF-10A breast epithelial cells, cell proliferation increased after insulin treatment by 180%. IR-phosphorylation, AKT-phosphorylation and ERK1/2 phosphorylation were all significantly increased while early apoptosis decreased after insulin treatment. Analysis of cell cycle however did not indicate a change in progression. Microarray analysis indicated that insulin treatment may increase expression of genes related to cancer growth. Leptin treatment increased cell proliferation and also increased ERK1/2-phosphorylation, while AKT-phosphorylation was not affected. Leptin did not change cell cycle progression. TNF-α treatment increased cell proliferation and also increased ERK1/2 phosphorylation, while AKT-phosphorylation was not changed. TNF-α treatment tended to increase apoptosis, the change however was not statistically significant. In SK-BR-3 breast cancer cells, cell proliferation did not change after insulin treatment. IR-phosphorylation and AKT-phosphorylation increased after insulin treatment, while ERK1/2-phosphorylation decreased. Gene expression of cyclin D and cyclin E increased with insulin treatment, while apoptotic rate and cell cycle profile were also not affected. Cell proliferation increased by 115% after treatment with 100 ng/ml leptin. ERK1/2-phosphorylation however decreased, while AKT-phosphorylation tended to increase, but the change was not statistically significant. Cell cycle profile was not affected by leptin treatment, G1-phase however tended to increase, but the change was again not statistically significant. Cell proliferation increased by 59% after 48 h treatment with 10 ng/ml TNF-α. AKT-phosphorylation and ERK1/2-phosphorylation increased with TNF-α treatment. Cell cycle analysis showed a decrease in S-phase and G2-phase, indicative of a decrease in cell cycle progression. These results indicate that none of the examined obesity-related factors is convincingly identified as the main molecular link between obesity and postmenopausal breast cancer. Conversely, all treatments affected each of the cell lines in, at least, one of the examined aspects. This indicates that many of the obesity-related factors may affect breast cancer and that a single breast tumour may utilise a unique combination of those factors to promote growth. All treatments increased proliferation in MCF-10A breast epithelial cells, with additional analysis generally supporting growth promotion. Insulin treatment particularly increased cell proliferation, while leptin and TNF-α increased MAP-kinase signalling. This may indicate that insulin and adipokines may have a higher impact on breast cancer aetiology than on breast cancer progression.

El adenocarcinoma ductal pancreático (PDAC) es la quinta causa de muerte por cáncer en los países desarrollados y uno de los tumores humanos más agresivos. A pesar del papel clave de la angiogénesis -la formación de nuevos vasos a partir de otros preexistentes- en la progresión y metástasis de muchos tumores, su papel en PDAC ha sido poco caracterizado. El activador tisular del plasminógeno (tPA), una proteína multifuncional que regula numerosas funciones celulares, ejerce efectos proangiogénicos en modelos in vivo de PDAC, aunque no se han analizado los mecanismos moleculares responsables de estos efectos. Esta tesis trata de identificar el papel de tPA en la angiogénesis del PDAC, así como de descubrir los factores responsables de la sobreexpresión de tPA en cáncer de páncreas. En primer lugar, hemos demostrado que los efectos pro-angiogénicos de tPA pueden ser tanto directos como indirectos. Por un lado, aunque tPA no cambia los niveles de moléculas pro-angiogénicas como VEGF, TGF-b, IL-1 o IL-8 producidas por las células tumorales o endoteliales, sí que induce la sobreexpresión y activación de MMP-9, una metaloproteasa implicada en promover angiogénesis, sugiriendo por tanto que esta proteína puede mediar de forma indirecta los efectos proangiogénicos de tPA. Por otro lado, hemos encontrado que tPA, de forma independiente de su actividad catalítica, promueve directamente la proliferación, migración y tubulogénesis de las células endoteliales. Estos efectos son mediados por la activación en estas células de las rutas de señalización ERK1/2, AKT y JNK. Además, mediante siRNA o inhibidores químicos, hemos encontrado que Annexina A2, Galectina-1 y EGFR son necesarios para la activación de la señalización inducida por tPA en células endoteliales. Finalmente, hemos visto que citoquinas inflamatorias e hipoxia, dos eventos asociados a PDAC y además inductores de angiogénesis, dan lugar a un fuerte incremento de los niveles de tPA en células tumorales pancreáticos. Todos estos datos apoyan un mecanismo de retroalimentación positiva entre estímulos proangiogénicos presentes en las células tumorales y el estroma, y el incremento de la molécula proangiogénica tPA. / Pancreatic ductal adenocarcinoma (PDAC) is the fifth leading cause of cancer death in the developed countries and one of the most aggressive human tumors. Despite the key contribution of angiogenesis – the growth of new vessels from pre-existing ones- to the progression and spread of many cancers, its role in PDAC has been poorly characterized. Tissue plasminogen activator (tPA), a multifunctional protein regulating a broad range of cellular functions, has been reported to exert pro-angiogenic effects in in vivo models of PDAC, although the underlying molecular mechanism has not been analyzed. This work aims to elucidate the role of tPA in the angiogenesis of PDAC as well as to identify the factors responsible for tPA increase in pancreatic cancer. First, we demonstrated that tPA pro-angiogenic effects are both indirect and direct. On the one hand, tPA does not change the levels of the pro-angiogenic molecules VEGF, TGF-b, IL-1 or IL-8 produced by pancreatic tumoral cells or endothelial cells, but it is involved in MMP-9 –a potent stimulator of angiogenesis- upregulation and activation in pancreatic and endothelial cells, suggesting that this matrix metalloproteinase can indirectly mediate tPA angiogenic effects. On the other hand, we found that tPA, in a catalytic-independent way, directly promotes endothelial cell proliferation, migration and tubulogenesis. These direct effects of tPA are mediated by activation of ERK1/2, AKT and JNK signaling pathways in endothelial cells. In addition, using siRNA technology or chemical inhibitors, we found that AnnexinA2, Galectin-1 and EGFR are required for tPA-mediated signaling activation in endothelium. Finally, we found that inflammatory cytokines and hypoxia, two hallmarks of PDAC and also angiogenic stimuli, lead to a sharp increase in tPA levels in pancreatic tumoral cells. These data support a feed-back loop between proangiogenic stimuli present in both tumoral and stromal cells and the increase of the proangiogenic molecule tPA.