As anti-cancer agents, retinoid induce cell growth arrest and differentiation, while HDACIs cause cell differentiation, growth inhibition, death and inhibit angiogenesis in many cancer types. However, a proportion of patients respond poorly to these therapies. My studies, presented here, aimed to improve the anti-cancer effects of these agents by identifying key factors which mediate cancer cell sensitivity or resistance to their action. In this study I have found that the clinically used retinoid, 13-cis RA, exerts its anti-cancer signal in a manner similar to atRA, by modulating the transcriptional response of retinoid-regulated genes. HDACI-induced cytotoxicity is significantly enhanced when combined with IFNα in 8 out of 9 cancer cell lines from various organ origins. Sensitivity to the combination treatment correlated with an absence of basal p21 protein expression, and cell cycle arrest. Knocking-down p21 gene expression further sensitized cancer cells to the combination therapy. Moreover, IFNα and HDACI co-operatively inhibited pro-angiogenic gene expression in cancer cells, and the combination therapy decreased endothelial cell migration, invasion, and capillary tubule formation. Further experiments on p21 as a resistance factor to anti-cancer treatment demonstrated that conditioned media from breast cancer MCF-7 cells transfected with p21 siRNA, induced significantly less endothelial cell migration, invasion and vascular sprouting, compared with media from cells transfected with scrambled siRNA. LC/MS analysis of the conditioned media revealed that Trx secretion was significantly reduced after p21 knockdown. The reduction in Trx secretion following p21 knockdown was due to a direct effect of p21 siRNA on the expression of intracellular TBP2 in neuroblastoma, prostate and lung cancer cells. Consistent with this result, media from MCF7 cells transfected with TBP2-specific siRNA alone, promoted endothelial cell invasion and vascular sprouting, Trx knockdown resulted in opposite effects, and the anti-angiogenic effect of p21 siRNA was offset by simultaneous TBP2 siRNA transfection. ChIP assay revealed that p21 directly bound to an E2F1-bindng site in the TBP2 gene promoter. These data indicate that p21 promoted tumour-driven angiogenesis through transcriptional repression of TBP2. Collectively, my experiments indicate several potential treatment targets directed toward enhancing the effectiveness of HDACIs and retinoids.

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Research on angiogenesis has been focused on developing anti-angiogenic therapies to target endothelial cell-specific signalling pathways, as a mean to limit tumour outgrowth and metastasis. One of the main targets is the endothelial cell-specific Tie2 receptor and its ligands, the angiopoietins, which controls the later stages of angiogenesis. Although the angiopoietin/Tie2 signalling pathways have been well characterized, the molecular mechanism by which the ligands regulate Tie2 activity remains unclear. To address this question, we determined whether the activation mechanism of Tie2 is induced by dimerisation alone, or whether subsequent relative rotation of the kinase domain is required. Here we employed a coiled-coiled based protein engineering approach to identify the relative orientations of the kinase domains that are optimal for Tie2 activation. By replacing the extracellular domain of Tie2 with the dimeric parallel coiled-coil motif Put3cc, we generated ligand-independent homodimers of the kinase domains Put3cc-Tie2 I-VII that have distinct orientations. We show that dimerisation is sufficient to induce Tie2 activation and downstream activation of Akt, and that varying the interface of the kinase domain in Tie2 dimers can increase its catalytic efficiency. In addition we examined for the presence of potential dimerisation within the transmembrane and intracellular domain of Tie2. We show that the KD and potentially the TM contain dimerisation motifs that stabilise Tie2 in the inactive and active conformations. In addition, we show that deletion of the potential coiled-coil motif in the JM does not disrupt dimerisation but decreases the catalytic efficiency of Tie2. Finally, we propose that the activation mechanism of Tie2 may be similar to the previously described asymmetric dimer formation of EGFR and FGFR receptors.

Orientador: Edgard Graner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-14T03:22:55Z (GMT). No. of bitstreams: 1 Seguin_Fabiana_M.pdf: 3111522 bytes, checksum: 0ecd0d27711d3b04c7d5e9c386bea758 (MD5) Previous issue date: 2009 / Resumo: A enzima ácido graxo sintase (FASN), cuja expressão e atividade estão elevadas em várias neoplasias malignas humanas, é responsável pela síntese endógena de ácidos graxos saturados de cadeia longa e consequentemente para a síntese de fosfolipídios de membrana. A inibição de FASN por orlistat (Xenical), uma droga anti-obesidade, é descrita como tendo propriedades anti-neoplásicas no câncer de próstata e mama e no melanoma, além de desempenhar um provável papel anti-angiogênico, uma vez que também inibe a proliferação de células endoteliais e a neovascularização em ensaio ex vivo. Em trabalho recente realizado por nosso grupo de pesquisa, foi demonstrado que o tratamento de camundongos portadores de melanomas intraperitoneais com orlistat reduziu em cerca de 50% o número de metástases para linfonodos mediastínicos. Em outro estudo, também realizado por nosso grupo, foi observado que a inibição da atividade de FASN pode ter um papel sobre a linfangiogênese induzida por melanomas experimentais, pois a densidade de vasos linfáticos ao redor destes tumores foi significantemente aumentada pelo tratamento com orlistat. Considerando o papel biológico aparentemente relevante da FASN na disseminação metastática de melanomas, o presente trabalho teve como objetivo principal investigar, em ensaio in vivo, o papel desta enzima no processo de angiogênese induzida por implantes intradérmicos de células de melanoma (B16F10) em camundongos (C57Bl6). Através de um microscópio de dissecção e da obtenção de imagens dos vasos sanguíneos peritumorais, a rede vascular foi avaliada com o auxílio do programa Scion Image. Foi observado que a densidade de vasos sanguíneos ao redor dos tumores tratados com orlistat foi significantemente reduzida em relação aos grupos controle (p=0,024; teste de Mann-Whitney). Além disso, os tumores foram medidos e seus volumes calculados, verificamos que o tratamento com orlistat não alterou significativamente o seu crescimento. Através de reações de RT-PCR semiquantitativo em amostras de RNA total extraído dos tumores, foi observado que a expressão dos mensageiros de FASN não foi alterada pelo tratamento com orlistat. Por outro lado, houve aumento da quantidade dos mensageiros para VEGFA nos tumores dos grupos tratados com orlistat. Através da construção de curvas de proliferação e de experimentos de citometria de fluxo, foram avaliados os efeitos do tratamento da linhagem celular derivada de endotélio de aorta de coelho (RAEC) com cerulenina e orlistat. A adição de cerulenina (0,70 µg/ml) e orlistat (100 µM) ao meio de cultura das células RAEC provocou significativa inibição do crescimento celular, em comparação com as células controle. Finalmente, a incubação das células RAEC com meio previamente condicionado pelas células de melanoma B16F10 provocou aumento na taxa de crescimento, confirmando o potencial angiogênico destas últimas. Em conjunto, estes achados sugerem que a inibição da atividade de FASN pode ter um papel na redução da angiogênese induzida por melanomas experimentais, sugerindo que o bloqueio de FASN possa ser um alvo em potencial para a terapia anti-angiogênica. / Abstract: The metabolic enzyme fatty acid synthase (FASN) is over expressed in many human malignancies, being responsible for the endogenous synthesis of longchain saturated fatty acids and consequently for the production of cell membrane phospholipids. Inhibition of FASN by orlistat (Xenical), an anti-obesity drug, has anti-neoplastic properties in prostate and breast cancer as well as in melanoma. In addition, FASN seems to participate in angiogenesis, since its blockage inhibits the proliferation of endothelial cells and neovascularization in an ex vivo assay. In a recent study conducted by our group, it was demonstrated that the treatment of mice bearing melanoma with orlistat was able to reduce by 50% the number of metastases to the mediastinal lymph nodes. We also observed that the inhibition of FASN activity may have a role in the melanoma-induced lymphangiogenesis, since the extension of lymphatic vessels around the tumors was significantly increased by the treatment with orlistat. Considering that the biological role of FASN is relevant in the metastatic spread of melanoma, the main goal of this study was to investigate the role of this enzyme in the angiogenesis process induced by intradermal implantation of melanoma cells (B16F10) in mice (C57Bl6). By using a microscope for dissection, we analyzed the images of the peritumoral blood vessels and the vascular network with the aid the Scion Image software. The blood vessel density around the tumors mice treated with orlistat decreased in comparison with the control group. Moreover, the tumors were measured and their volumes calculated. And no statistically significant changes observed. The effect of orlistat or cerulenin on the proliferation of a cell line derived from the rabbit aorta endothelium (RAEC) was analyzed by the construction of growth curves and flow cytometry. The addition of cerulenin (0.70 µg/ml) or orlistat (100 µM) to the culture medium of RAEC cells caused significant inhibition of cell growth compared with the non-treated cells. Taken together, these findings suggest that inhibition of FASN activity may have a role in the reduction of angiogenesis induced by experimental melanomas, suggesting that FASN could be a potential target for the anti-angiogenic therapy. / Mestrado / Patologia / Mestre em Estomatopatologia

Objective: In this study, I have directly tracked the progression of angiogenesis for three different types of breast cancer cell lines; MDA-MB-231, MCF-7, and MDA-MB-468. Each of these cell lines is known to overexpress different receptors, which may affect a tumor’s growth rate and perhaps its ability to undergo angiogenesis. Here, I measure and compare the growth, extent, and time of onset for angiogenesis. Methods: I used SCID mice to profile each of the different breast cancer cell lines. The growth rate of each tumor, along with its blood vessel development, was monitored and imaged using lipid-coated microbubbles and contrast-enhanced ultrasound (CEUS). A Vevo 2100 pre-clinical ultrasound machine was used for the imaging experiments. To track development of angiogenesis, mice were injected with perfluorobutane gas microbubbles of 1-2 microns diameter. Bubble perfusion into the tumor is an indicator of the presence of blood vessel formation. A custom image analysis program was developed in Matlab™ to eliminate breathing artifacts and track microbubble motion based on their high temporal frequency signature ("flicker"). Results: My experiments demonstrated that, although different cell lines grow at different rates, microbubbles begin to penetrate the tumor when it reaches approximately a size of approximately 3 mm in diameter. Therefore, the onset of angiogenesis occurred at different times (MCF-7 occurring first at around 9 days, MDA-MB-468 occuring at 12 days post inoculation, and MDA-MB-231 occurring at 17 days post tumor cell inoculation). Matlab™ analysis demonstrates consistent angiogenic behavior among the three cell lines. Conclusion: For all cell lines, angiogenesis started when the volume of the tumor was approximately 21.76 mm³, consistent with previous studies. As angiogenesis progressed, there was a drop in tumor blood flow. This can be explained by the sudden influx of oxygen when angiogenesis first begins. This momentarily inhibits new blood vessel formation while the tumor continues to steadily grow. After this sudden drop, tumor vascularization resumes a steady increase.

Ischemic stroke related brain damage causes loss of multiple cell types, including neural and vascular cells. The extent of post-stroke neurogenesis and angiogenesis predicts the level of functional regeneration/recovery after stroke. In this regard, my thesis was focused on defining the molecular process that modulates post-stroke functional recovery by co-ordinating post-stroke neurovascular remodeling. Since stroke-related brain damage releases enriched local microenvironmental cues, I examined the role of a signaling-induced epigenetic pathway, an atypical protein kinase C (aPKC)-mediated phosphorylation of CREB Binding Protein (CBP), in regulating post-stroke neurovascular remodeling and functional recovery. This pathway has previously been shown to be activated by metformin, an adenosine monophosphate kinase (AMPK) activator, to promote the differentiation of neural precursors in the developing and adult brain. Here, I first developed a murine focal cortical ischemic stroke model with persistent motor function deficits by combined intra-cortical injections of endothelin-1 (ET-1) and L-NAME into the sensorimotor cortex. Second, I applied the ET-1/L-Name-induced focal cortical stroke model in a knock-in mouse CBPS436A where the aPKC-CBP pathway is deficient, and showed that the aPKC-CBP pathway is involved in post-stroke functional recovery by coordinating neurovascular remodeling. Specifically, CBPS436A-KI mice displayed reduced motor recovery, correlated with reduced vascular remodeling and impaired post-stroke angiogenesis. Intriguingly, I also observed that CBPS436A-KI mice showed a reduction in the population of stroke-induced newborn pericytes but an increase in the population of perivascularly-derived neural precursors, implying that the aPKC-CBP pathway may be involved in the process that reprograms pericytes into neural precursors. Together, this study elucidates the novel role of the aPKC-CBP pathway in modulating neurovascular remodeling and functional recovery following focal ischemic cortical stroke.

Angiogenesis, the formation of new capillaries from preexisting vessels, plays an essential role in revascularization of the myocardium following myocardial infarction (MI). Interleukin-1β (IL-1β), a proinflammatory cytokine increased in the heart following MI, is shown to be essential for angiogenesis in the invasiveness of tumor cells, the progression of arthritic conditions and endometriosis, and the promotion of wound healing. Here we studied the steps of angiogenesis in response to IL-1β in cardiac microvascular endothelial cells (CMECs) and aortic tissue. Cell cycle progression analysis using flow cytometry indicated a G0/G1 phase cell cycle arrest in IL-1β-stimulated cells. IL-1β significantly reduced levels of fibrillar actin in the cytoskeleton, a pre-requisite for tube formation, as indicated by phalloidin-FITC staining. Wound healing assays demonstrated IL-1β prevents cell-to-cell contact formation. On the other hand, vascular endothelial growth factor-D (VEGF-D) initiated restoration of the cell monolayer. IL-1β significantly inhibited in vitro tube formation as analyzed by three-dimensional collagen matrix assay. Aortic ring assay demonstrated that IL-1β inhibits basal and VEGF-D-stimulated microvessel sprouting from aortic rings. The data presented here are novel and of significant interest, providing evidence that IL-1β impedes the process of angiogenesis in myocardial endothelial cells.