As proteínas S100A8/A9 são expressas em diferentes tipos celulares e quando sozinhas ou complexadas e em baixas concentrações, promoveram proliferação, migração celular e formação de estruturas capilares. Por outro lado, quando em altas concentrações, esse complexo inibe o crescimento de diversos tipos de células tumorais murinas e humanas. Ainda, tanto a proteína S100A9 humana, quanto um peptídeo sintético idêntico a porção C-terminal da proteína S100A9 murina (pS100A9m) possuem efeitos antinociceptivo e imunorregulatório. Apesar dessas evidencias, até o momento não foi investigado o efeito do pS100A9m sobre a angiogênese e a tumorigênese. Portanto, o objetivo do presente estudo foi investigar, in vitro, o efeito do pS100A9m sobre os eventos fundamentais envolvidos com a angiogênese e o desenvolvimento tumoral. Para tanto, a fim de avaliar o efeito do pS100A9m sobre a angiogênese foi utilizada a linhagem de células endoteliais tímicas murinas (tEnd.1) nos ensaios de proliferação, migração da célula endotelial em meio de cultura, avaliada nos modelos de wound healing e transwell ou migração em meio condicionado, obtido de células tumorais LLC WRC256, avaliada no modelo de transwell, ensaio de adesão (aos componentes de matriz, tais como o colágeno tipo I, fibronectina e laminina) e formação de tubos em matrigel tridimensional (3D). Para os estudos sobre o efeito do pS100A9m sobre as células tumorais, foi utilizada a linhagem de células LLC WRC256 para realização dos ensaios funcionais de proliferação, migração (wound healing) e adesão (sobre os componentes da matriz extracelular). Os resultados obtidos demonstraram que o pS100A9m inibe a proliferação, migração, adesão sobre os componentes de matriz e, consequentemente, a formação de estruturas capilares em matriz 3D. Em relação às células tumorais LLC WRC256, foi observada, novamente, a ação inibitória do pS100A9m sobre os eventos de proliferação e migração. Em relação à adesão, o peptídeo aumentou a capacidade de adesão das células tumorais sobre o colágeno tipo I e fibronectina, porém inibiu a adesão dessas células sobre laminina. Em conclusão, os dados aqui obtidos demonstram que o pS100A9m inibe in vitro os eventos fundamentais envolvidos com a angiogênese e com a progressão tumoral. Desta forma, o peptídeo da porção C-terminal da proteína S100A9 pode ser considerado uma nova ferramenta para o estudo da angiogênese e tumorigênese, além apresentar potencial para uma possível aplicação terapêutica nesses processos / The S100A8/A9 proteins are expressed in different cell types and alone or when complexed, and at low concentrations promoted proliferation, cell migration and formation of capillary structures. On the other hand, at higher concentrations, this compound inhibits the growth of many types of murine and human tumor cells. Moreover, both human S100A9 protein and a synthetic peptide identical to the C-terminal portion of murine S100A9 (mS100A9p) present antinociceptive and immunomodulatory effects. Despite these evidences, the effect of mS100A9p on angiogenesis and tumorigenesis has not been investigated. Therefore, the aim of this study was to investigate the in vitro effect of mS100A9p on crucial events involved in angiogenesis and tumor development. For this, in order to evaluate the effect of mS100A9p on angiogenesis was used the murine endothelial cell line derived from thymus hemangioma (tEnd.1) for proliferation assays, endothelial cell migration in the presence of culture medium (scratch wound healing and chemotaxis assays) or in conditioned medium prevenient from LLC WRC256 tumor cells (chemotaxis assays), adhesion assay (on extracellular matrix components, such as type I collagen, fibronectin and laminin) and tube like-structure formation in 3D matrix. For the analyzes of the effect of mS100A9p on tumor cells, the cell line LLC WRC256 was used to perform functional assays such as proliferation, migration (scratch wound healing model) and adhesion (on components of the extracellular matrix). The results showed that the mS100A9p inhibits the proliferation, migration and adhesion of endothelial cells to the matrix components and consequently the formation of capillary structures in 3D matrix. Regarding LLC WRC256 tumor cells, it was observed again the inhibitory action of the mS100A9p on proliferation and migration events. In relation to cellular adhesion, this peptide increased this parameter of tumor cells on type I collagen and fibronectin. However mS100A9p inhibited the adhesion of these cells on laminin. In conclusion, the data obtained show that the mS100A9p inhibits in vitro crucial events involved in angiogenesis and tumor progression. Thus, the C-terminal portion of murine S100A9 protein may be considered as a new tool for the study of tumorigenesis and angiogenesis besides presenting potential to a possible therapeutic application in these processes

The acquisition and maintenance of cell division potential are important characteristics of tumorigenesis. Human telomerase reverse transcriptase (TERT) and telomerase RNA (TR) can immortalize cells through telomere maintenance, and telomerase activity is one factor that contributes to the in vitro transformation of normal cells. In vitro and in vivo evidence suggest that telomerase maintains telomeres as a functional multimer. In addition, hTERT may possess telomere maintenance-independent functions. To examine the effects of hTERT loss upon an in vitro generated tumorigenic cell line we created a tumorigenic cell line from human embryonic kidney cells through expression of the SV40 early region, H-RasG12V and a Cre-mediated excisable hTERT. These immortalized cells exhibited robust anchorage-independent colony growth and tumor formation in immuno-deficient mice. Cre recombinase expression resulted in the excision of hTERT from the tumorigenic cell lines, restoring cell mortality. A return to immortality was conferred by the re-introduction of wild-type hTERT, but not with hTERT point mutations in the N-terminus (hTEN) and reverse transcriptase (RT) domains that impair in vitro telomere elongation activity. The onset of cell mortality was not immediate, and the hTERT-excised tumorigenic cells exhibited clonal variation in the anchorage-independent colony growth assay and upon tumor formation in immuno-deficient mice. We hypothesized that tumorigenic potential was not related strictly to hTERT presence, but rather telomere length and/or integrity. To investigate this possibility we maintained the tumorigenic cell lines in the continuous presence of hTERT to permit telomere elongation prior to hTERT excision; subsequently, after hTERT excision tumor formation persisted, thus demonstrating a dependence on telomere length and not hTERT presence per se. To investigate the functional multimerization of hTERT in vivo we tested if two defective hTERT polypeptides with mutations in the hTEN and RT domains could restore telomere elongation activity in vivo. Unfortunately, during the course of these experiments an unanticipated recombination event occurred that restored a catalytically active hTERT transgene. Further in vivo analysis of hTERT multimerization should be carefully designed, accounting for the selective survival advantage bestowed by wild-type hTERT. This study provides compelling evidence that hTERT does not possess telomere maintenance-independent functions.

The acquisition and maintenance of cell division potential are important characteristics of tumorigenesis. Human telomerase reverse transcriptase (TERT) and telomerase RNA (TR) can immortalize cells through telomere maintenance, and telomerase activity is one factor that contributes to the in vitro transformation of normal cells. In vitro and in vivo evidence suggest that telomerase maintains telomeres as a functional multimer. In addition, hTERT may possess telomere maintenance-independent functions. To examine the effects of hTERT loss upon an in vitro generated tumorigenic cell line we created a tumorigenic cell line from human embryonic kidney cells through expression of the SV40 early region, H-RasG12V and a Cre-mediated excisable hTERT. These immortalized cells exhibited robust anchorage-independent colony growth and tumor formation in immuno-deficient mice. Cre recombinase expression resulted in the excision of hTERT from the tumorigenic cell lines, restoring cell mortality. A return to immortality was conferred by the re-introduction of wild-type hTERT, but not with hTERT point mutations in the N-terminus (hTEN) and reverse transcriptase (RT) domains that impair in vitro telomere elongation activity. The onset of cell mortality was not immediate, and the hTERT-excised tumorigenic cells exhibited clonal variation in the anchorage-independent colony growth assay and upon tumor formation in immuno-deficient mice. We hypothesized that tumorigenic potential was not related strictly to hTERT presence, but rather telomere length and/or integrity. To investigate this possibility we maintained the tumorigenic cell lines in the continuous presence of hTERT to permit telomere elongation prior to hTERT excision; subsequently, after hTERT excision tumor formation persisted, thus demonstrating a dependence on telomere length and not hTERT presence per se. To investigate the functional multimerization of hTERT in vivo we tested if two defective hTERT polypeptides with mutations in the hTEN and RT domains could restore telomere elongation activity in vivo. Unfortunately, during the course of these experiments an unanticipated recombination event occurred that restored a catalytically active hTERT transgene. Further in vivo analysis of hTERT multimerization should be carefully designed, accounting for the selective survival advantage bestowed by wild-type hTERT. This study provides compelling evidence that hTERT does not possess telomere maintenance-independent functions.

<p>Abstract</p><p>The trefoil factor family of secreted proteins contains three members; trefoil factor 1 or TFF1, trefoil factor 2 or TFF2, and trefoil factor 3 or TFF3. These three proteins share a conserved 42-43 amino acid domain containing 6 cysteine residues resulting in three disulfide bonds that holds the protein in a characteristic three-loop or "trefoil structure" known as the P domain. TFF1 is primarily localized to the stomach and secreted by the gastric mucosa while TFF2 and TFF3 are primarily localized to the colon and duodenum and secreted by the goblet cells. All three of these proteins play a protective role in the gastrointestinal tract where they are normally localized and have been identified as possible tumor suppressors, however, these proteins are also upregulated in cancer within tissues where they are not normally expressed including the breast, pancreas, prostate, and liver. The mechanisms by which two of these factors, TFF1 and TFF3, promote tumorigenesis remain largely undefined. In this dissertation we will attempt to elucidate these mechanisms as well as the regulation of these two proteins in both pancreatic and prostate cancer. Many of the underlying genetic and molecular mechanisms involved in the development of both pancreatic and prostate cancer remain largely unknown and as a result, therapeutic and diagnostic tools for treating these diseases are not as effective as they could be. By deciphering the role of TFF1 and TFF3 in these cancers, they could potentially serve as new therapeutic targets or biomarkers for treating both diseases.</p><p>Chapter 2 of this dissertation will examine the functional role of TFF1 promoting tumorigenesis in pancreatic and prostate cancer. We will show that TFF1 expression is critical for the viability of both pancreatic and prostate cancer cells and that reduction of TFF1 expression in these cells results in decreased tumorigenicity when implanted in immunocompromised mice. It will also be demonstrated that TFF1's function in promoting tumorigenicity is its ability to assist tumor cells overcome the tumor suppressive barrier of senescence. Thirdly, we show that the form of senescence that TFF1 assists in allowing the cells overcome is oncogene-induced senescence (OIS). Lastly, a cell cycle array identifies the potential downstream target p21CIP, a cyclin-dependent kinase inhibitor and OIS marker, whose expression is induced by loss of TFF1 expression.</p><p>In Chapter 3 of this work, we examine the role of another trefoil factor family member, TFF3, and its role in promoting prostate tumorigenesis. Just as with TFF1, it appears that TFF3 3 expression is critical for prostate cancer cell viability and tumorigenicity using the same experimental techniques used in Chapter 2. Using a genetically defined model of prostate cancer, a PI3-kinase-dependent regulatory mechanism of TFF3 emerges in this prostate cancer context. Using this system we begin to see a divergence in both regulation and function of TFF1 and TFF3 in prostate cancer. Finally, a mouse model expressing TFF3 was developed to monitor the histopthological changes associated with expression of this protein. Initial characterization of this model suggests a hyperplastic phenotype coinciding with TFF3 expression in the prostate.</p><p>The two studies in this dissertation establish a role of TFF1 and TFF3 in both prostate and pancreatic tumorigenesis and demonstrate that ablation of expression of both proteins is a potent inhibitor of tumorigenesis. With this knowledge, it is possible that TFF1 and TFF3 may become a potential therapeutic target or diagnostic marker for better treatment of prostate and pancreatic cancer.</p> / Dissertation

The Reelin gene located on the human chromosome region 7q22, encodes an extracellular matrix glycoprotein, a ligand for ApoER2 and low-density lipoprotein receptors (LDL) Receptor, is required for mediating the correct positioning of neurons during embryonic brain development1. In the current study, first we applied RT-PCR and immunohistochemistry analysis (IHC) analysis on tissue microarrays (TMA) to verify the Reelin expression patterns in a variety of adult tissues, suggesting additional roles for Reelin in stabling the cyto-architecture and controlling the remodeling of many organs during development. Second, we report the Reelin expression status in tumorigenesis. We discover that the loss of Reelin expression is associated with multiple types of cancers, including more than 80% of both breast and colorectal cancers. Interestingly, our study also found suspension small cell lung cancer (SCLC) cell lines that grow as large aggregates retained high Reelin expression, whereas attached non small cell lung cancer cultures do not. That may imply the Reelin expression may be also associated with cell culture morphology and growth characteristics in the in vitro culture system for lung cancers. Our results here also demonstrated that epigenetic silencing of Reelin expression by DNA hypermethylation in tumors directly correlates with loss of Reelin expression in many cancers. Reelinmethylation was reversed and expression restored by treating tumor cell lines with the demethylating agent 5-aza-2-deoxycytidine. In conclusion, from the molecular basis of Reelingene inactivation in human cancer here, we propose that the Reelinvariation in more than 80% of breast and colorectal cancers makes it a significant novel tumor marker.

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression during melanoma carcinogenesis remains unclear. In this study, adding exogenous HDGF stimulated the invasion and colonies formation of B16-F10 melanoma cells. Adenovirus vectors encoding HDGF and HDGF-RNAi were generated and characterized to up- and down-regulated HDGF expression in B16-F10 melanoma cells. It was found that HDGF overexpression stimulated the proliferation, invasiveness, anchorage-independent growth of B16-F10 melanoma cells whereas HDGF knockdown exerted opposite effects. In lung-metastasis model, intravenous injection of HDGF-overexpressing melanoma cells resulted in increased metastasis while HDGF-downregulated melanoma cells caused decreased metastasis. Similarly, in primary melanoma model, subcutaneous injection of HDGF-overexpressing melanoma cells enhanced while HDGF-downregulated melanoma cells reduced the tumor burden in mice. Histological analysis revealed increased tumor proliferation and neovascularization with concomitant reduction of apoptosis in HDGF-overexpressing melanoma. Moreover, HDGF-overexpressing melanoma also exhibited enhanced propensity to metastasize from the primary tumors to lymph node and lung. Finally, it was found that HDGF overexpression increased nuclear factor kappa B (NF£eB) activities and Akt phosphorylation up and down stream alternation like PI3K, PTEN, I£eB and it¡¦s subunit IKK£\, IKK£], IKK£^ in melanoma cells. It also found that HDGF overexpression influenced MITF and HIF1£\ in melanoma after gene delivery. HDGF also altered EMT changes like E,N-cadherin, vimentin, and £],£^-catenin. The present study provides conclusive evidence that HDGF upregulation promotes the growth and metastasis of melanoma by promoting the survival and vascularization. Besides, HDGF knockdown may constitute a novel strategy for melanoma control.

Der hochkonservierte Wnt/ß-Catenin-Signaltransduktionsweg spielt eine wichtige Rolle während der Embryonalentwicklung, der Homöostase und der Tumorgenese in Adulten. Die BCL9 Proteine wurden zunächst als Kofaktoren dieses Signalweges identifiziert. Entsprechend agiert BCL9/Legless als essentieller Wnt/ß-Catenin-Kofaktor in Drosophila. Jedoch scheint die Rolle von BCL9 und BCL9-2, der Orthologe von Legless, in Vertebraten komplexer zu sein. Des Weiteren wurden die genauen Funktionen der BCL9 Proteine während der intestinalen Homöostase und Tumorgenese bislang wenig untersucht. Es konnte jedoch bereits gezeigt werden, dass BCL9-2 in intestinalen und Mammakarzinomen verstärkt expremiert wird. Diese Arbeit beschreibt erstmalig den Einfluss und die Funktion von BCL9-2 während der intestinalen Tumorgenese. BCL9-2 beinflusst die Tumorprogression positiv durch Verstärkung des Wnt/ß-Catenin-Signaltransduktionsweg und der Expression von Zielgenen, die Tumorwachstum und -invasion vermitteln. Zudem aktiviert BCL9-2 die Transkription von ß-Catenin-unabhängigen Genen durch einen neuartigen Mechanismus. Im Gegensatz zu BCL9, welches in allen humanen und murinen intestinalen Zelltypen expremiert wurde, beschränkte sich die BCL9-2 Expression auf die Zotten des Darmes. Die Wnt/ß-Catenin-positiven Krypten hingegen zeigten keinerlei BCL9-2-Expression auf, was darauf hinweist, dass BCL9-2 für den Wnt/ß-Catenin-Signalweg bei der intestinalen Homöostase entbehrlich ist. Während jedoch BCL9 Proteinlevel in Kolontumoren, im Vergleich zum normalen Epithel, unverändert blieben, wurde BCL9-2 bereits in frühen Stadien der Tumorgenese und in 90% aller Kolonkarzinome stark expremiert. Darüber hinaus führte transgene Überexpression von BCL9-2 im Darm von K19-BCL9-2;APCMin/+ Mäusen zu einer verstärkten Formation von Adenomen, deren Invasion und einem verringerten Überleben der Versuchstiere. Wie anhand von TOP/FOP Luciferase Reportergen-Versuchen gezeigt werden konnte, korrelierte die Stärke der BCL9-2-Proteinexpression mit der Aktivität des Wnt/ß-Catenin-Signalweges in Kolonkarzinomzellen. Zudem regulierte BCL9-2 die Transkription einiger ß-Catenin-abhängiger und darüber hinaus ß-Catenin-unabhängiger Zielgene, die bei der Tumorentstehung eine wichtige Rolle spielen. Des Weiteren zeigt diese Arbeit, dass in Kolonkarzinomzellen die BCL9-2 abhängige Transkription von CDX1 und CDX2 durch SP1-bindende Elemente über deren proximale Promotoren vermittelt wurde. Mittels Immunpräzipitation konnte zudem eine Interaktion zwischen BCL9-2 und SP1 in Kolonkarzinomzellen bestätigt werden. Zusammenfassend zeigt diese Arbeit, dass BCL9-2-Überexpression in frühen Phasen der intestinalen Tumorgenese die Progression von benignen Tumoren in invasive Karzinome fördert. Diese Eigenschaft wird durch verschiedene Mechanismen vermittelt: Zum einen verstärkt BCL9-2 die Expression einiger Wnt/ß-Catenin-abhängiger Zielgene; zum anderen reguliert BCL9-2 ß-Catenin-unabhängige Gene, die für die Tumorgenese eine wichtige Rolle spielen. Diese Funktion wird vermutlich durch die Bindung an SP1 Transkriptionsfaktoren und damit an die Promotoren von BCL9-2 Zielgenen vermittelt, was zu der verstärkten Expression von Genen führt, die die Tumorprogression und Invasion fördern.

Multiple endocrine neoplasia syndrome type 1 (MEN1) is an autosomal dominantly inherited disease, which is described as an association of tumors mainly in endocrine organs, including pancreas and adrenal glands. Pancreatic neuroendocrine tumors (PNETs) are the most common cause of death in MEN1 patients. More than one third of the MEN1 patients also develop enlargement of the adrenals. MEN1 is caused by a germline mutation of MEN1 gene, a tumor suppressor gene that is located on the human chromosome 11. As noticed, the MEN1 related tumors often develop prior to inactivation of both wild type alleles, indicating MEN1 haploinsufficiency. In this thesis, I utilized a conventional Men1 mouse model that has the phenotype mimicking the human MEN 1 traits, in order to investigate MEN1 tumorigenesis in endocrine pancreas and adrenal glands.   The microvascular aberrations contributing to development and maintenance of PNETs were characterized. The increased vascular density of PNETs developed in the Men1 mice was paralleled by an early and extensive redistribution of pericytes within endocrine tissue. These morphological alterations were supported by fine-tuned variations in expression of several angiogenic regulators  (VEGF, FGF and PDGF) and were further potentiated by hypoxia. Vascular reactivity and blood perfusion of tumor arterioles were significantly altered in response to glucose and L-nitro-arginine methyl ester. Investigation of adrenals from10-month-old Men1 mice showed 681 proteins in mass spectrometry data sets, in which 52 proteins were commonly found in the Men1+/+ and Men1+/- adrenals, and the differential expression between the genotypes reached significant levels. Prdx3, catalyzing the reduction of oxidative stress to cell survival, is one of the overexpressed proteins. Some proteins belonging to the PPARα pathway, e.g. ACLY were also overexpressed. Subsequent microRNA (miRNA) profiling analysis of adrenals from the same age group revealed 31 miRNAs whose expression was significantly altered in comparison between the genotypes. The tumor suppressor miRNAs, miR-486, miR-330 and miR-214, were significantly downregulated in Men1+/- adrenals. The latter, miR-214, is known to inhibit ACLY expression. This finding was in concordance with the proteomic analysis. The oncogene miRNAs, miR-132 and miR-494, were significantly enhanced in the Men1+/- adrenals. Gene ontology analysis demonstrated overrepresentation of the miRNA-targeted genes that are involved in nucleic acid metabolism, vasculature development, angiogenesis, and transcription. Together, these finding after validation in humans may be exploited to improve MEN1 cancer treatment.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate the expression of mRNA targets, and are aberrantly expressed in several cancers, including breast cancer. Using a transgenic mouse model of mammary tumorigenesis (MTB-IGFIR), a miRNA array was previously performed in our lab to study the expression level of various miRNAs in mammary tumours compared to wild-type mammary tissue. Next, the expression of a number of the differentially expressed miRNAs was confirmed and manipulated in a tumour cell line (RM11A) generated from a MTB-IGFIR mammary tumour. Synthetic miRNA precursors and inhibitors were then used to overexpress and knockdown, respectively, the levels of five miRNAs: miR-31, miR-183, miR-200c, miR-210, and miR-378. Upon optimization of miRNA overexpression and downregulation in RM11A cells, this study tested the effects of these miRNAs on cellular growth, survival, or invasiveness in vitro. Compared to negative controls, overexpression of all five miRNAs was associated with a significant decrease in cellular invasion, while only the overexpression of miR-31 had a significant effect on proliferation. No significant effects were found on cell survival. Our results implicate these five miRNAs in different aspects of mammary tumorigenesis, as well as having a tissue specific role in RM11A cells.

No description available.